RESUMEN

Soybean hairy roots transformed with the resveratrol synthase and resveratrol oxymethyl transferase genes driven by constitutive Arabidopsis actin and CsVMV promoters were characterized. Transformed hairy roots accumulated glycoside conjugates of the stilbenic compound resveratrol and the related compound pterostilbene, which are normally not synthesized by soybean plants. Expression of the non-native stilbenic phytoalexin synthesis in soybean hairy roots increased their resistance to the soybean pathogen Rhizoctonia solani. The expression of the AhRS3 gene resulted in 20% to 50% decreased root necrosis compared to that of untransformed hairy roots. The expression of two genes, the AhRS3 and ROMT, required for pterostilbene synthesis in soybean, resulted in significantly lower root necrosis (ranging from 0% to 7%) in transgenic roots than in untransformed hairy roots that had about 84% necrosis. Overexpression of the soybean prenyltransferase (dimethylallyltransferase) G4DT gene in soybean hairy roots increased accumulation of the native phytoalexin glyceollin resulting in decreased root necrosis.

Asunto(s)

Glycine max/inmunología , Glycine max/metabolismo , Inmunidad de la Planta/fisiología , Plantas Modificadas Genéticamente/inmunología , Plantas Modificadas Genéticamente/metabolismo , Sesquiterpenos/metabolismo , Dimetilaliltranstransferasa , Raíces de Plantas/genética , Raíces de Plantas/inmunología , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/microbiología , Rhizoctonia/patogenicidad , Glycine max/genética , Glycine max/microbiología , Fitoalexinas

RESUMEN

A nonantibiotic/herbicide-resistance selection system for plastid transformation is described here in technical detail. This system is based on the feedback-insensitive anthranilate synthase (AS) α-subunit gene of tobacco (ASA2) as a selective marker and tryptophan (Trp) or indole analogs as selection agents. AS catalyzes the first reaction in the Trp biosynthetic pathway, naturally compartmentalized in the plastids, by converting chorismate to anthranilate and is subjected to feedback inhibition by Trp. In addition to Trp, various Trp analogs and indole compounds that can be converted to Trp analogs can also inhibit AS activity and therefore are toxic to cells. When cells are made to express the feedback-insensitive ASA2, they acquire resistance to these analogs and can be selected for during transformation process. We have demonstrated the feasibility of this selection system in tobacco (Nicotiana tabacum L. cv. Petit Havana). ASA2-expressing transplastomic plants were obtained on medium supplemented with either 7-methyl-DL-tryptophan (7-MT) or 4-methylindole (4-MI). These plants show normal phenotype and fertility and transmit the resistance to the selection agents strictly maternally.

Asunto(s)

Antranilato Sintasa/genética , Cloroplastos/genética , Indoles/metabolismo , Nicotiana/genética , Triptófano/metabolismo , Antranilato Sintasa/antagonistas & inhibidores , Células Cultivadas , Regulación de la Expresión Génica de las Plantas , Técnicas de Transferencia de Gen , Vectores Genéticos/biosíntesis , Plantas Modificadas Genéticamente/genética , Subunidades de Proteína/genética , Plantones/crecimiento & desarrollo , Transformación Genética , Triptófano/análogos & derivados , Triptófano/biosíntesis

RESUMEN

The effects of resveratrol and pterostilbene on in vitro growth of three soybean pathogens were tested to determine whether these stilbenic compounds could potentially be targets to increase innate resistance in transgenic soybean plants. Growth of Macrophomina phaseolina, Rhizoctonia solani, and Sclerotinia sclerotiorum was measured on solid and in liquid media amended with resveratrol and pterostilbene (concentration in the media of resveratrol at 100 µg/ml and pterostilbene at 25 µg/ml). All three fungi were very sensitive to pterostilbene in potato dextrose agar (PDA), which reduced colony area of each of the three pathogens to less than half of the control 3 days after incubation. The three fungal pathogens were less sensitive to resveratrol compared with pterostilbene; however, area under the curve (AUC) calculated from colony areas measured over 3 days was significantly (P < 0.05) less than the control for S. sclerotiorum and R. solani on PDA with resveratrol or pterostilbene. AUC for M. phaseolina on PDA with pterostilbene was significantly (P < 0.05) lower than the control whereas, on PDA with resveratrol, AUC for M. phaseolina was lower than the control but the difference was nonsignificant (P > 0.05). AUC for all three fungi was significantly lower (P < 0.05) on PDA with pterostilbene than with resveratrol. In potato dextrose broth (PDB) shake cultures, AUC for all three fungi was significantly (P < 0.01) lower in pterostilbene than in the control. AUC for R. solani and S. sclerotiorum was significantly lower (P < 0.01) in resveratrol than the control, whereas AUC for M. phaseolina in resveratrol was lower, but not significantly (P > 0.05) different from the control. AUC in pterostilbene was highly significantly (P < 0.01) lower than in resveratrol for M. phaseolina and significantly (P < 0.05) lower for R. solani but the difference for S. sclerotiorum was nonsignificant (P > 0.05). There was a trend for lower mass accumulation of all three fungi in either pterostilbene or resveratrol compared with the control during the course of the experiment; however, S. sclerotiorum appeared to recover from the effects of pterostilbene between days 2 and 4. Results of biochemical analyses of the PDB over time indicated that the three fungi degraded resveratrol, with nearly 75% reduction in concentration in M. phaseolina, 80% in S. sclerotiorum, and 60% in R. solani PDB cultures by day 4 of fungal growth. M. phaseolina and S. sclerotiorum were able to resume growth after early inhibition by resveratrol after its concentration was reduced in the cultures through degradation, whereas R. solani was less efficient in resveratrol degradation and was not able to overcome its inhibitory effects on growth. The capacity to degrade pterostilbene was lowest in M. phaseolina compared with S. sclerotiorum and R. solani and the recovery of M. phaseolina cultures after initial growth inhibition by pterostilbene was minimal. The potential products of resveratrol and pterostilbene degradation by fungi were identified to be dimers and various oxidation products.

Asunto(s)

Ascomicetos/crecimiento & desarrollo , Glycine max/microbiología , Enfermedades de las Plantas/microbiología , Rhizoctonia/crecimiento & desarrollo , Estilbenos/farmacología , Ascomicetos/efectos de los fármacos , Medios de Cultivo , Resveratrol , Rhizoctonia/efectos de los fármacos , Estilbenos/química , Estilbenos/metabolismo

RESUMEN

The response of soybean transgenic plants, with suppressed synthesis of isoflavones, and nontransgenic plants to two common soybean pathogens, Macrophomina phaseolina and Phytophthora sojae, was studied. Transgenic soybean plants of one line used in this study were previously generated via bombardment of embryogenic cultures with the phenylalanine ammonia lyase, chalcone synthase, and isoflavone synthase (IFS2) genes in sense orientation driven by the cotyledon-preferable lectin promoter (to turn genes on in cotyledons), while plants of another line were newly produced using the IFS2 gene in sense orientation driven by the Cassava vein mosaic virus constitutive promoter (to turn genes on in all plant parts). Nearly complete inhibition of isoflavone synthesis was found in the cotyledons of young seedlings of transgenic plants transformed with the IFS2 transgene driven by the cotyledon-preferable lectin promoter compared with the untransformed control during the 10-day observation period, with the precursors of isoflavone synthesis being accumulated in the cotyledons of transgenic plants. These results indicated that the lectin promoter could be active not only during seed development but also during seed germination. Downregulation of isoflavone synthesis only in the seed or in the whole soybean plant caused a strong inhibition of the pathogen-inducible glyceollin in cotyledons after inoculation with P. sojae, which resulted in increased susceptibility of the cotyledons of both transgenic lines to this pathogen compared with inoculated cotyledons of untransformed plants. When stems were inoculated with M. phaseolina, suppression of glyceollin synthesis was found only in stems of transgenic plants expressing the transgene driven by a constitutive promoter, which developed more severe infection. These results provide further evidence that rapid glyceollin accumulation during infection contributes to the innate soybean defense response.

Asunto(s)

Glycine max , Phytophthora , Ascomicetos/genética , Regulación de la Expresión Génica de las Plantas , Phytophthora/genética , Enfermedades de las Plantas/genética , Plantas Modificadas Genéticamente , Glycine max/genética

RESUMEN

Metabolic changes were studied, which accompanied the conversion of 6month old HiII maize non-regenerable (NR) calli into regenerable (R) calli when cultured for 63days with 10% polyethylene glycol (PEG) (3350MW) in culture medium. The conversion of 6month old NR to R callus morphotype caused by PEG application decreased cell wall contents in callus dry mass and changed cell wall phenolics making their profile similar to that of R callus by reduction of lignin and ester- and ether-bound phenolic concentrations, including p-coumaric acid and ester- and ether-bound diferulates and by increase of the ratios of ester- and ether-bound ferulic acid/coumaric acid and ferulic acid/diferulic acid in cell walls of NR callus. Some similar changes of cell wall phenolics caused by PEG application were also found in 48month old NR callus, that changed the morphology, but did not regenerate plants. However, there were no changes in the old callus in levels of total ester and ether-bound cell wall phenolics and substantially smaller decreases were found in ratios of ester- and ether-bound ferulic acid/coumaric acid and ferulic acid/diferulic acid, as well as in diferulate concentrations compared to young NR callus cultured with PEG. Remarkably, application of PEG also changed the primary metabolism of young NR callus tissues, so that they acquired metabolic features of highly regenerable callus. These data clearly suggest that PEG alters metabolism of NR calli, so they acquire biochemical characteristics of R calli, and that adaptive osmotic adjustments vary in different types of callus tissues.

Asunto(s)

Metaboloma/efectos de los fármacos , Polietilenglicoles/farmacología , Zea mays/citología , Zea mays/efectos de los fármacos , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Fenol/metabolismo , Factores de Tiempo , Zea mays/metabolismo

RESUMEN

Plants recognize invading pathogens and respond biochemically to prevent invasion or inhibit colonization in plant cells. Enhancing this response in crop plants could improve sustainable methods to manage plant diseases. To enhance disease resistance in soybean, the soybean phytoalexin glyceollin was assessed in soybean hairy roots of two soybean genotypes, Spencer and PI 567374, transformed with either soybean isoflavone synthase (IFS2) or chalcone synthase (CHS6) genes that were inoculated with the soybean pathogens Diaporthe phaseolorum var. meridionales, Macrophomina phaseolina, Sclerotinia sclerotiorum, and Phytophthora sojae. The hairy-root-transformed lines had several-fold decreased levels of isoflavone daidzein, the precursor of glyceollin, and considerably lower concentrations of glyceollin induced by pathogens measured 5 days after fungal inoculation compared with the nontransformed controls without phenolic transgenes. M. phaseolina, P. sojae, and S. sclerotiorum grew much more on IFS2- and CHS6-transformed roots than on control roots, although there was no significant difference in growth of D. phaseolorum var. meridionales on the transformed hairy-root lines. In addition, glyceollin concentration was lower in D. phaseolorum var. meridionales-inoculated transformed and control roots than roots inoculated with the other pathogens. Glyceollin inhibited the growth of D. phaseolorum var. meridionales, M. phaseolina, P. sojae, S. sclerotiorum, and three additional soybean pathogens: Cercospora sojina, Phialophora gregata, and Rhizoctonia solani. The most common product of glyceollin conversion or degradation by the pathogens, with the exception of P. sojae, which had no glyceollin degradation products found in the culture medium, was 7-hydroxyglyceollin.

Asunto(s)

Glycine max/inmunología , Glycine max/microbiología , Enfermedades de las Plantas/inmunología , Pterocarpanos/farmacología , Estructura Molecular , Enfermedades de las Plantas/microbiología , Raíces de Plantas/microbiología , Pterocarpanos/química

RESUMEN

When Zea mays callus cultures of two different genotypes were treated with the osmoticum mannitol (0.53M) for 24h their ability to reduce the tetrazolium derivative 2,3,5-triphenyltetrazolium chloride (TTC) to form the insoluble red compound formazan is stimulated. The formazan can be extracted with 95% ethanol for quantitation and this reaction has been used as a measure of viability since only live cells can carry out this reduction. In order to determine the cause of the increased TTC reduction caused by mannitol we carried out metabolic profiling analysis using GC-MS to identify 80 compounds. There were increases in sugar alcohols, hexoses except fructose and in total sugars. The total organic acid pools did not change and nitrogen containing compounds decreased slightly. Principle component analysis showed a large treatment effect due to changes in carbohydrate and nitrogen metabolism. These results indicate that the increased carbohydrate available for the citric acid cycle may be the cause of the increased TTC reduction observed after the mannitol treatment.

Asunto(s)

Manitol/farmacología , Metaboloma , Sales de Tetrazolio/metabolismo , Zea mays/anatomía & histología , Zea mays/efectos de los fármacos , Metaboloma/efectos de los fármacos , Modelos Biológicos , Oxidación-Reducción/efectos de los fármacos , Análisis de Componente Principal , Zea mays/metabolismo

RESUMEN

The yeast N-acetyltransferase MPR1 gene has previously been shown to confer resistance to the toxic proline analogue azetidine-2-carboxylic acid (A2C) in yeast and transgenic tobacco. Here experiments were carried out to determine if MPR1 and A2C can work as a selectable marker system for plant transformation. The MPR1 gene was inserted into a binary vector under the control of the cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and transformed into tobacco via the Agrobacterium tumefaciens-mediated leaf disc method. A2C was applied in the selection medium to select for putative transformants. PCR analysis showed that 28.4% and 66.7% of the plantlets selected by 250 muM and 300 muM A2C were positive for the MPR1 gene, respectively. Southern and northern blot analysis and enzyme activity assay confirmed the stable gene incorporation, transcription, and translation of the MPR1 transgene in the transgenic plants. The transgene-carrying T(1) progeny could be distinguished from the recessive progeny when grown on 400, 450, or 500 muM A2C. Examination of the metabolism of 22 transgenic plants by gas chromatography-mass spectrometry profiling did not reveal any significant changes. In conclusion, the results demonstrate that MPR1/A2C is a safe and efficient selection system that does not involve microbial antibiotic or herbicide resistance genes. Recent studies showed that MPR1 can protect yeast against oxidative stresses by decreasing the accumulation of the proline catabolite Delta(1)-pyrroline-5-carboxylate (P5C). However, H(2)O(2) treatment resulted in contradictory responses among the five transgenic lines tested. Further experiments are required to assess the response of MPR1 transgenic plants under oxidative stress.

Asunto(s)

Acetiltransferasas/genética , Ácido Azetidinocarboxílico/farmacología , Nicotiana/genética , Prolina/análogos & derivados , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Selección Genética , Transformación Genética/efectos de los fármacos , Ácido Azetidinocarboxílico/química , Segregación Cromosómica/genética , Cruzamientos Genéticos , Cromatografía de Gases y Espectrometría de Masas , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes Fúngicos/genética , Marcadores Genéticos , Vectores Genéticos , Genoma de Planta/genética , Peróxido de Hidrógeno/farmacología , Kanamicina/farmacología , Metaboloma/genética , Estrés Oxidativo/efectos de los fármacos , Plantas Modificadas Genéticamente , Análisis de Componente Principal , Prolina/química , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Transgenes/genética

RESUMEN

ABSTRACT Understanding the metabolic responses of the plant to a devastating foliar disease, soybean rust, caused by Phakopsora pachyrhizi, will assist in development of cultivars resistant to soybean rust. In this study, differences in phenolic metabolism were analyzed between inoculated and noninoculated plants using two susceptible and three resistant soybean genotypes with known resistance genes. Rust infection resulted in increased accumulation of isoflavonoids and flavonoids in leaves of all soybean genotypes tested. Although the soybean phytoalexin glyceollin was not detected in leaves of uninfected plants, accumulation of this compound at marked levels occurred in rust-infected leaves, being substantially higher in genotypes with a red-brown resistant reaction. In addition, there was inhibition of P. pachyrhizi spore germination by glyceollin, formononetin, quercetin, and kaempferol. However, there was no correlation between concentrations of flavonoids quercetin and kaempferol and rust-induced isoflavonoid formononetin in soybean leaves and rust resistance. Lignin synthesis also increased in all inoculated soybean genotypes whereas there was no significant difference in all noninoculated soybean genotypes. Cell wall lignification was markedly higher in inoculated resistant lines compared with inoculated susceptible lines, indicating a possible protective role of lignin in rust infection development.

Asunto(s)

Basidiomycota/efectos de los fármacos , Basidiomycota/crecimiento & desarrollo , Flavonoles/metabolismo , Glycine max/metabolismo , Glycine max/microbiología , Enfermedades de las Plantas/microbiología , Flavonoles/química , Flavonoles/farmacología , Flavonoles/fisiología , Genotipo , Inmunidad Innata/genética , Inmunidad Innata/fisiología , Isoflavonas/química , Isoflavonas/metabolismo , Isoflavonas/farmacología , Isoflavonas/fisiología , Quempferoles/química , Quempferoles/metabolismo , Quempferoles/farmacología , Quempferoles/fisiología , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Pterocarpanos/química , Pterocarpanos/metabolismo , Pterocarpanos/farmacología , Pterocarpanos/fisiología , Quercetina/química , Quercetina/metabolismo , Quercetina/farmacología , Quercetina/fisiología , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/crecimiento & desarrollo

RESUMEN

To modify the level and composition of isoflavones, the important bioactive constituents of soybean seeds, soybean was transformed via co-bombardment of embryogenic cultures with three DNA cassettes containing the CHS6-chalcone synthase and IFS2-isoflavone synthase genes, and a fragment of PAL5-phenylalanine ammonia-lyase gene, all in sense orientation under the lectin promoter mixed with the selectable marker gene, HPT (hygromycin phosphotransferase) under the 35S promoter. Four of six fertile lines produced integrated all four genes. Isoflavone levels were lower in T1 mature seeds of 5 of the 6 lines compared to the control. Transgene segregation was found in one selected line, with formation of additional sublines with different transgene composition found also in the homozygous plants. Decreased isoflavone concentrations (by about 70%) were found in T4 homozygous seeds of the two lines studied in detail here. The embryo axes accumulated most of the glycitein and contained a higher isoflavone concentration than the cotyledons. Expression of transgenes driven by the lectin promoter reduced the isoflavone concentration only in the cotyledons and not in embryo axes, indicating that this promoter is preferably active in cotyledons.

Asunto(s)

Glycine max/genética , Glycine max/metabolismo , Isoflavonas/química , Fenol/química , Semillas/metabolismo , Southern Blotting , Cromatografía Líquida de Alta Presión , Silenciador del Gen , Homocigoto , Isoflavonas/metabolismo , Modelos Biológicos , Modelos Genéticos , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transgenes

RESUMEN

Genetic engineering of chloroplasts normally requires the stable introduction of bacterial derived antibiotic or herbicide-resistance genes as selective markers. Ecological and health concerns have been raised due to the presence of such genes within the environment or the food supply. One way to overcome this issue is the use of plant genes able to confer a metabolic or developmental advantage to the transformed cells manipulating the plant's biosynthetic pathways. We explored the feasibility of using, for plastid transformation, the selection system based on the feedback-insensitive anthranilate synthase (AS) alpha-subunit gene of tobacco (ASA2) as a new selective marker and the indole analogue 4-methylindole (4MI) or the tryptophan analogue 7-methyl-DL-tryptophan (7MT) as the selection agents. An expression cassette containing Prrn-ASA2 was effectively integrated into the region between accD and ycf4 of the tobacco plastome by the biolistic process. Plastid transgenic plants were obtained on medium supplemented with 300 microM 7MT or 4MI. Transplastomic plants showed normal phenotype and fertility and the resistance to the selection agents 7MT and 4MI was transmitted maternally. The plastid transformed lines also exhibited a higher level of AS enzyme activity that was less sensitive to Trp-feedback inhibition and, consequently, increased free Trp levels in leaves about 7-fold.

Asunto(s)

Antranilato Sintasa/metabolismo , Nicotiana/genética , Proteínas de Plantas/metabolismo , Plastidios/genética , Transformación Genética , Antranilato Sintasa/genética , Proteínas de Plantas/genética , Plastidios/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Nicotiana/metabolismo , Triptófano/metabolismo

RESUMEN

Effective selectable markers are needed for basic research and commercial applications that do not involve antibiotic or herbicide resistance. A novel selection system based on a feedback-insensitive anthranilate synthase alpha-subunit of tobacco (ASA2) as selectable marker using either 4-methylindole (4MI) or 7-methyl-DL-tryptophan (7MT) as the selection agent was developed. We found that these two components were able to discriminate better between ASA2 expressing and untransformed lines than the most commonly used analog 5-methyltryptopan (5MT) in the seedling growth inhibition test. We successfully integrated an expression cassette containing an ASA2 cDNA driven by a cauliflower mosaic virus 35S promoter into tobacco leaf discs by A. tumefaciens and selected transgenic plants on medium supplemented with 300 microM of 7MT or 4MI. Due to the expression of the feedback-insensitive ASA2, the transgenic lines produced showed higher free tryptophan (Trp) concentrations than the untransformed WT control. These results demonstrate the feasibility of the selection system with the ASA2 gene in combination with the use of Trp or indole analogs as selective agent.

Asunto(s)

Antranilato Sintasa/genética , Indoles/farmacología , Nicotiana/genética , Triptófano/farmacología , Agrobacterium tumefaciens/genética , Antranilato Sintasa/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Vectores Genéticos/genética , Indoles/química , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Plantones/efectos de los fármacos , Plantones/genética , Plantones/crecimiento & desarrollo , Nicotiana/efectos de los fármacos , Nicotiana/crecimiento & desarrollo , Transformación Genética/efectos de los fármacos , Triptófano/química

RESUMEN

Soybean [Glycine max (L.) Merr.] embryogenic cultures were transformed by particle bombardment with the feedback-insensitive tobacco anthranilate synthase (AS) gene ASA2 driven by the CaMV 35S promoter and selected using hph as the selectable marker gene. Only one of eight regenerated lines that set seed and contained ASA2 expressed the gene highly and contained increased free tryptophan (Trp) levels in leaves, seeds and embryogenic cultures. Leaf extracts of the ASA2 expressing line contained about twice as much AS enzyme activity as the untransformed control and this activity was only slightly more feedback-insensitive. Amino acid analysis showed that both leaves and embryogenic tissue cultures of the ASA2 expressing line had four to five-times the normal levels of free Trp and slightly higher free tyrosine and phenylalanine. The seed total Trp content was only slightly increased. Metabolic profiling-analysis by GC-MS detected no other consistent differences. These studies show that the ASA2 gene can be expressed in soybean and that modest changes in Trp synthesis occurs.

Asunto(s)

Antranilato Sintasa/genética , Antranilato Sintasa/metabolismo , Glycine max/metabolismo , Nicotiana/enzimología , Triptófano/metabolismo , Núcleo Celular , Plantas Modificadas Genéticamente , Glycine max/genética , Nicotiana/genética , Transformación Genética

RESUMEN

Twelve independent lines were transformed by particle bombardment of soybean embryogenic suspension cultures with the tobacco anthranilate synthase (ASA2) promoter driving the uidA (beta-glucuronidase, GUS) reporter gene. ASA2 appears to be expressed in a tissue culture specific manner in tobacco (Song H-S, Brotherton JE, Gonzales RA, Widholm JM. Tissue culture specific expression of a naturally occurring tobacco feedback-insensitive anthranilate synthase. Plant Physiol 1998;117:533-43). The transgenic lines also contained the hygromycin phosphotransferase (hpt) gene and were selected using hygromycin. All the selected cultures or the embryos that were induced from these cultures expressed GUS measured histochemically. However, no histochemical GUS expression could be found in leaves, stems, roots, pods and root nodules of the plants formed from the embryos and their progeny. Pollen from some of the plants and immature and mature seeds and embryogenic cultures initiated from immature cotyledons did show GUS activity. Quantitative 4-methylumbelliferyl-glucuronide (MUG) assays of the GUS activity in various tissues showed that all with observable histochemical GUS activity contained easily measurable activities and leaves and stems that showed no observable histochemical GUS staining did contain very low but measurable MUG activity above that of the untransformed control but orders of magnitude lower than the constitutive 35S-uidA controls used. Low but clearly above background levels of boiling sensitive GUS activity could be observed in the untransformed control immature seeds and embryogenic cultures using the MUG assay. Thus in soybean the ASA2 promoter drives readily observable GUS expression in tissue cultures, pollen and seeds, with only extremely low levels seen in vegetative tissues of the plants. The ASA2 driven expression seen in mature seed was, however, much lower than that seen with the constitutive 35S promoter; less than 2% in seed coats and less than 0.13% in cotyledons and embryo axes. The predominate tissue culture specific expression pattern of the ASA2 promoter may be useful for genetic transformation of crops.

Asunto(s)

Antranilato Sintasa/genética , Glucuronidasa/análisis , Glycine max/genética , Nicotiana/genética , Plantas Modificadas Genéticamente/metabolismo , Antranilato Sintasa/análisis , Antranilato Sintasa/metabolismo , Flores/metabolismo , Genes Reporteros , Hojas de la Planta/metabolismo , Tallos de la Planta/metabolismo , Plantas Modificadas Genéticamente/embriología , Plásmidos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/análisis , Semillas/metabolismo , Glycine max/embriología , Técnicas de Cultivo de Tejidos , Transformación Genética

RESUMEN

Metabolite profiling of untransformed and cyanamide hydratase- (Cah) transformed (denoted 1C) soybean (Glycine max [L.] Merrill) leaves revealed only small differences in plants grown in the greenhouse or in the dark for 24 h, indicating that the Cah enzyme that converts cyanamide to urea has no substrates in soybean leaves and does not affect metabolism. Untransformed leaves sprayed with 0.5% cyanamide developed necrotic lesions within 2 h in the light but not in the dark. The sprayed 1C leaves showed little visible damage and accumulated high concentrations of urea, amino acids, and some sugars, but sucrose decreased over a 24 h period. The untransformed necrotic leaves also accumulated some urea and amino acids apparently due to cyanamide degradation, while sucrose and some organic acids decreased. Sprayed 1C leaves in the dark for 24 h contained very little urea and lower sugar levels. The untransformed sprayed leaves accumulated some organic acids, some sugars including sucrose, and urea and total amino acids. Unsprayed plants of both lines placed in the dark for 24 h showed increases in some amino acids and phosphate, and decreases in other amino acids, sugars, and organic acids. Thus the Cah enzyme can detoxify cyanamide by conversion to urea that is converted to amino acids. Other metabolic changes associated with leaf necrosis and darkness are also described. Principal component analysis confirmed the similarities and differences observed. Comparison of the GC-MS metabolic profiling analysis of amino acids with a dedicated system shows large differences, indicating a limitation of the former system.

Asunto(s)

Cianamida , Oscuridad , Glycine max/metabolismo , Hidroliasas/metabolismo , Hojas de la Planta/metabolismo , Aminoácidos/metabolismo , Hidroliasas/genética , Glycine max/enzimología , Glycine max/genética

RESUMEN

To facilitate future investigations of glyphosate-resistance mechanisms, three approaches were taken to obtain Arabidopsis thaliana variants that differed in glyphosate response. Recurrent selection by spraying with sub-lethal glyphosate concentrations was performed with Columbia-0 seedlings. After seven cycles of treatment, no resistance was found. A population of 800,000 ethylmethanesulfonate-mutagenized M(2) seedlings was screened on agar containing 0.2mM glyphosate, a lower concentration than that previously used in other studies, and no resistant mutants were recovered. Seventy-two Arabidopsis ecotypes were screened with glyphosate and a range of responses was observed. In a follow-up experiment on a subset of these ecotypes, reduction of seed yield by 11.5 g/ha glyphosate (about 1% the typical field use rate) ranged among ecotypes from 0% to >90%, relative to untreated controls. However, even the least sensitive ecotypes were severely injured by relatively low glyphosate rates. Overall, attempts to select Arabidopsis seedlings that were significantly glyphosate-resistant were unsuccessful and consistent with previous reports. Arabidopsis ecotypes identified with differential glyphosate responses could be used for further studies though the inherently high sensitivity of Arabidopsis to glyphosate could limit their utility in studying glyphosate-resistance mechanisms.

Asunto(s)

Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Glicina/análogos & derivados , Resistencia a los Herbicidas/genética , Herbicidas/farmacología , Medios de Cultivo , Relación Dosis-Respuesta a Droga , Glicina/farmacología , Mutación , Selección Genética , Glifosato

RESUMEN

Soybean hairy roots, transformed with the soybean chalcone synthase (CHS6) or isoflavone synthase (IFS2) genes, with dramatically decreased capacity to synthesize isoflavones were produced to determine what effects these changes would have on susceptibility to a fungal pathogen. The isoflavone and coumestrol concentrations were decreased by about 90% in most lines apparently due to gene silencing. The IFS2 transformed lines had very low IFS enzyme activity in microsomal fractions as measured by the conversion of naringenin to genistein. The CHS6 lines with decreased isoflavone concentrations had 5 to 20-fold lower CHS enzyme activities than the appropriate controls. Both IFS2 and CHS transformed lines accumulated higher concentrations of both soluble and cell wall bound phenolic acids compared to controls with higher levels found in the CHS6 lines indicating alterations in the lignin biosynthetic branch of the pathway. Induction of the soybean phytoalexin glyceollin, of which the precursor is the isoflavone daidzein, by the fungal pathogen Fusarium solani f. sp. glycines (FSG) that causes soybean sudden death syndrome (SDS) showed that the low isoflavone transformed lines did not accumulate glyceollin while the control lines did. The (iso)liquritigenin content increased upon FSG induction in the IFS2 transformed roots indicating that the pathway reactions before this point can control isoflavonoid synthesis. The lowest fungal growth rate on hairy roots was found on the FSG partially resistant control roots followed by the SDS sensitive control roots and the low isoflavone transformants. The results indicate the importance of phytoalexin synthesis in root resistance to the pathogen.

Asunto(s)

Aciltransferasas/metabolismo , Glycine max/metabolismo , Oxigenasas/metabolismo , Raíces de Plantas/metabolismo , Aciltransferasas/genética , Cromatografía Líquida de Alta Presión , Regulación hacia Abajo , Hongos/crecimiento & desarrollo , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Estructura Molecular , Oxigenasas/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Raíces de Plantas/genética , Raíces de Plantas/microbiología , Plantas Modificadas Genéticamente , Propanoles/química , Propanoles/metabolismo , Glycine max/genética , Glycine max/microbiología , Espectrometría de Masa por Ionización de Electrospray

RESUMEN

The polyphenolic composition of 62 wild and weedy Mexican bean collections from diverse origins, grouped by their seed coat color, was assessed. According to spectrophotometric analysis, the range of total phenols, condensed tannins, and total anthocyanins presented wide differences. Furthermore, the phenolic acid, flavonoid, and anthocyanin profiles were analyzed using HPLC. Ferulic was the main phenolic acid. Kaempferol and quercetin were the main flavonoids, and the isoflavones daidzein and coumestrol were found in only low levels in few collections. Delphinidin was the main anthocyanidin found, followed by petunidin, cyanidin, malvidin, pelargonidin, and peonidin. The wide variation observed in polyphenolic contents was more related to their genotype than to the color factor. These results show that some wild and weedy beans are good sources of phenolic compounds for use in breeding programs focused on nutrition and health.

Asunto(s)

Flavonoides/análisis , Phaseolus/química , Fenoles/análisis , Semillas/química , Antocianinas/análisis , Cromatografía Líquida de Alta Presión , Pigmentación , Polifenoles , Espectrofotometría , Taninos/análisis

RESUMEN

Common bean effects on health have been related to its dietary fiber content and other active compounds. This study assessed the content of flavonoids, coumestrol, phenolic acids, galactooligosaccharides, and phytic acid in wild and cultivated Mexican common bean seeds (raw and cooked) and that of flavonoids, coumestrol, and phenolic acids in germinated bean seeds. The presence of isoflavones in raw bean seeds was not confirmed by the UV spectra. Quercetin, kaempferol, p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, and vanillic acid mean contents were 10.9, 52.3, 10.1, 9.6, 5.4, and 18.2 microg/g, respectively; raffinose, stachyose, verbascose, and phytic acid mean contents were 8.5, 56.3, 5.5, and 11.5 mg/g, respectively, in raw seeds. All compounds were affected by autoclaving, and germination resulted in a de novo synthesis of flavonols, phytoestrogens, and phenolic acids. The impact on health of common bean seed is affected by dietary burden, specific compounds content, and processing. On the other hand, germinated bean seed or beans sprouts may be sources of antioxidants and phytoestrogens.

Asunto(s)

Antioxidantes/análisis , Dieta , Promoción de la Salud , Phaseolus/química , Fitoestrógenos/análisis , Semillas/química , Flavonoides/análisis , Germinación , Isoflavonas/análisis , México , Oligosacáridos/análisis , Ácido Fítico/análisis , Semillas/crecimiento & desarrollo

RESUMEN

Embryogenic tissue cultures of soybean were transformed by particle bombardment with a vector pCHZ-II that carries the coding sequence for cyanamide hydratase (Cah), an enzyme that converts toxic cyanamide to urea, from the soil fungus Myrothecium verrucaria. The Cah gene was driven by the constitutive Arabidopsis thaliana actin-2 promoter and terminated with its cognate terminator. This vector also carries the hygromycin phosphotransferase gene (hpt) driven by the potato (Solanum tuberosum) ubiquitin-3 promoter. Twelve individual lines of transgenic plants that were obtained under hygromycin selection expressed Cah mRNA and exhibited resistance to hygromycin in leaf tissue culture, while the untransformed tissues were sensitive. Cah enzyme activity was present in extracts of transformed leaves and embryogenic tissue cultures when measured by a colorimetric assay and the presence of the Cah protein was confirmed by enzyme-linked immunosorbent assay (ELISA). Cah expression detoxified cyanamide in leaf callus and embryogenic cultures as well as in whole plants as shown by cyanamide resistance. The Cah-expressing plants grew and set seeds normally indicating that the Cah enzyme activity did not affect soybean plant metabolism. We also describe a test whereby callus was formed on cultured leaf tissue in the presence of hygromycin or cyanamide only if the hpt or Cah gene was expressed, respectively. This test is a convenient and cost-effective way to follow the marker gene in the primary regenerated plants and subsequent generations, which is particularly reliable for the hpt gene expression using hygromycin.

Asunto(s)

Cianamida/metabolismo , Hongos/enzimología , Glycine max/genética , Hidroliasas/genética , Cianamida/farmacología , Expresión Génica , Marcadores Genéticos , Hidroliasas/metabolismo , Inactivación Metabólica , Plantas Modificadas Genéticamente , Glycine max/enzimología