Regulation of plant immunity through modulation of phytoalexin synthesis.

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.

Tryptophan and indole analog mediated plastid transformation.

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.

Inhibitory effects of stilbenes on the growth of three soybean pathogens in culture.

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.

Glyceollin is an important component of soybean plant defense against Phytophthora sojae and Macrophomina phaseolina.

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.

Polyethylene glycol treatment promotes metabolic events associated with maize callus morphogenic competence.

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.

Response of soybean pathogens to glyceollin.

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.

Metabolic profiling to determine the cause of the increased triphenyltetrazolium chloride reduction in mannitol-treated maize callus.

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.

The application of the yeast N-acetyltransferase MPR1 gene and the proline analogue L-azetidine-2-carboxylic acid as a selectable marker system for plant transformation.

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.

The importance of phenolic metabolism to limit the growth of Phakopsora pachyrhizi.

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.

Tobacco plastid transformation using the feedback-insensitive anthranilate synthase [alpha]-subunit of tobacco (ASA2) as a new selectable marker.

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.

Modification of isoflavones in soybean seeds via expression of multiple phenolic biosynthetic genes.

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.

Effect of the expression of cyanamide hydratase on metabolites in cyanamide-treated soybean plants kept in the light or dark.

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.

Use of 4-methylindole or 7-methyl-DL-tryptophan in a transformant selection system based on the feedback-insensitive anthranilate synthase alpha-subunit of tobacco (ASA2).

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.

Expression of a feedback insensitive anthranilate synthase gene from tobacco increases free tryptophan in soybean plants.

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.

Specificity of expression of the GUS reporter gene (uidA) driven by the tobacco ASA2 promoter in soybean plants and tissue cultures.

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.

Identification of Arabidopsis thaliana variants with differential glyphosate responses.

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.

Modification of phenolic metabolism in soybean hairy roots through down regulation of chalcone synthase or isoflavone synthase.

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.

Polyphenols in wild and weedy Mexican common beans (Phaseolus vulgaris L.).

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.

Chemical components with health implications in wild and cultivated Mexican common bean seeds (Phaseolus vulgaris L.).

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.

Expression of a fungal cyanamide hydratase in transgenic soybean detoxifies cyanamide in tissue culture and in planta to provide cyanamide resistance.

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.

Isoflavonoid accumulation in soybean hairy roots upon treatment with Fusarium solani.

Hairy roots were initiated from two soybean [Glycine max (L.) Merr.] genotypes with different susceptibility (susceptible 'Spencer' and partially resistant 'PI567.

Osmotic induced stimulation of the reduction of the viability dye 2,3,5-triphenyltetrazolium chloride by maize roots and callus cultures.

Live cells can reduce colorless 2,3,5-triphenyltetrazolium chloride (TTC) to a red insoluble compound, formazan. Maize (Zea mays) callus, when osmotically stressed by 0.53 mol/L mannitol, produced 7-times or more formazan than untreated control callus.

Length of time in tissue culture can affect the selected glyphosate resistance mechanism.

Usually, stepwise selection of plant suspension cultures with gradually increasing concentrations of the herbicide glyphosate results in the amplification of the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.

Complementation of the nuclear antisense rbcS-induced photosynthesis deficiency by introducing an rbcS gene into the tobacco plastid genome.

The small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a nuclear gene-encoded protein that is imported into chloroplasts where it assembles with the large subunit (LS) after removal of the transit peptide to form Rubisco. We have explored the possibility that the severe deficiency in photosynthesis exhibited in nuclear transgenic tobacco (line alpha5) expressing antisense rbcS coding DNA that results in low SS and Rubisco protein content [Rodermel et al. (1988) Cell 55: 673] could be complemented by introducing a copy of the rbcS gene into its plastid genome through chloroplast transformation.

Glyphosate selection of gene amplification in suspension cultures of 3 plant species.

Stepwise selection was carried out with increasing glyphosate concentrations to produce suspension cultures of Medicago sativa L. (alfalfa), Glycine max L. (Merr.