Although promoter regions for many plant nuclear genes have been sequenced, identification of the active promoter sequence has been carried out only for the octopine synthase promoter. That analysis was of callus tissue and made use of an enzyme assay. We have analysed the effects of 5' deletions in a plant viral promoter in tobacco callus as well as in regenerated plants, including different plant tissues. We assayed the RNA transcription product which allows a more direct assessment of deletion effects. The cauliflower mosaic virus (CaMV) 35S promoter provides a model plant nuclear promoter system, as its double-strand DNA genome is transcribed by host nuclear RNA polymerase II from a CaMV minichromosome. Sequences extending to -46 were sufficient for accurate transcription initiation whereas the region between -46 and -105 increased greatly the level of transcription. The 35S promoter showed no tissue-specificity of expression.

Isoflavones have drawn much attention because of their benefits to human health. These compounds, which are produced almost exclusively in legumes, have natural roles in plant defense and root nodulation. Isoflavone synthase catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway. To identify the gene encoding this enzyme, we used a yeast expression assay to screen soybean ESTs encoding cytochrome P450 proteins. We identified two soybean genes encoding isoflavone synthase, and used them to isolate homologous genes from other leguminous species including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine, as well as from the nonleguminous sugarbeet. We expressed soybean isoflavone synthase in Arabidopsis thaliana, which led to production of the isoflavone genistein in this nonlegume plant. Identification of the isoflavone synthase gene should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes.

No abstract

Metabolic engineering for production of isoflavones in non-legume plants may provide the health benefits of these phytoestrogens from consumption of more widely used grains. In legumes, isoflavones function in both the symbiotic relationship with rhizobial bacteria and the plant defense response. Expression of a soybean isoflavone synthase (IFS) gene in Arabidopsis plants was previously shown to result in the synthesis and accumulation of the isoflavone genistein in leaf and stem tissue (Jung et al., 2000). Here we further investigate the ability of the heterologous IFS enzyme to interact with the endogenous phenylpropanoid pathway, which provides the substrate for IFS, and produces genistein in several plant tissue systems. In tobacco (Nicotiana tabacum) floral tissue that synthesizes anthocyanins, genistein production was increased relative to leaves. Induction of the flavonoid/anthocyanin branch of the phenylpropanoid pathway through UV-B treatment also enhanced genistein production in Arabidopsis. In a monocot cell system, introduced expression of a transcription factor regulating genes of the anthocyanin pathway was effective in conferring the ability to produce genistein in the presence of the IFS gene. Introduction of a third gene, chalcone reductase, provided the ability to synthesize an additional substrate of IFS resulting in production of the isoflavone daidzein in this system. The genistein produced in tobacco, Arabidopsis, and maize (Zea mays) cells was present in conjugated forms, indicating that endogenous enzymes were capable of recognizing genistein as a substrate. This study provides insight into requirements for metabolic engineering for isoflavone production in non-legume dicot and monocot tissues.

The plant genome responds to the bacteriophage P1-derived loxP-Cre site-specific recombination system. Recombination took place at loxP sites stably integrated in the tobacco genome, indicating that the Cre recombinase protein, expressed by a chimeric gene also stably resident in the genome, was able to enter the nucleus and to locate a specific 34 bp DNA sequence. An excisional recombination event was monitored by the acquisition of kanamycin resistance, which resulted from the loss of a polyadenylation signal sequence that interrupted a chimeric neomycin phosphotransferase II gene. Molecular analysis confirmed that the excision had occurred. Recombination occurred when plants with the integrated loxP construction were stably re-transformed with a chimeric cre gene and when plants with the introduced loxP construction were cross-bred with those carrying the chimeric cre gene. As assayed phenotypically, site-specific recombination could be detected in 50%-100% of the plants containing both elements of the system. Kanamycin resistance was detected at 2-3 weeks after re-transformation and in the first leaf of hybrid seedlings. This demonstration of the effectiveness of the loxP-Cre system in plants provides the basis for development of this system for such purposes as directing site-specific integration and regulation of gene expression.

The Sfreptomyces griseolus gene encoding herbicide-metabolizing cytochrome P450sul (CYP105Al) was expressed in transgenic tobacco (Nicofiana tabacum). Because this P450 can be reduced by plant chloroplast ferredoxin in vitro, chloroplast-targeted and nontargeted expression were compared. Whereas P450sul antigen was found in the transgenic plants regardless of the targeting, only those with chloroplast-directed enzyme performed P450sul-mediated N-dealkylation of the sulfonylurea 2-methylethyl-2,3-dihydro-N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl~-1,2-benzoisothiazole-7-sulfonamide-1,l-dioxide (R7402). Chloroplast targeting appears to be essential for the bacterial P450 to function in the plant. Because the R7402 metabolite has greater phytotoxicity than R7402 itself, plants bearing active P450sul are susceptible to injury from R7402 treatment that is harmless to plants without P45OSul. Thus, P450sul expression and R7402 treatment can be used as a negative selection system in plants. Furthermore, expression of P450sul from a tissue-specific promoter can sequester production of the phytotoxic R7402 metabolite to a single plant tissue. In tobacco expressing P450sul from a tapetum-specific promoter, treatment of immature flower buds with R7402 caused dramatically lowered pollen viability. Such treatment could be the basis for a chemical hybridizing agent.P450 monooxygenases play a central role in plant response to a variety of environmental challenges, including herbicide and other pesticide treatments (Cole, 1983;Durst, 1991). The best examples of this role are those P450s that can chemically alter an herbicide to a form with reduced phytotoxicity, resulting in much higher herbicide tolerance in the plant where this metabolism occurs (Frear et al., 1969(Frear et al., ,1991Sweetser et al., 1982; Jacobson and Shimabukuru, 1984;Brown, 1990). Significant variability in P450 enzyme activity exists among plant species, both in the presence of distinct enzymic forms and in their range of substrate specificity. This variability is a major determinant in herbicide selectivity, the differential herbicide sensitivity of weed and crop species. Altering herbicide selectivity, particularly confemng resistance in a crop plant, might be accomplished by expression of Present address:

No abstract

We have introduced chimeric genes containing polyadenylation signals from a human gene and two animal virus genes into tobacco cells. We see, in all three cases, inefficient and 'aberrant' utilization of the foreign polyadenylation signals. We find that a chimeric gene carrying the polyadenylation site of the human growth hormone gene is polyadenylated at three sites in the vicinity of the site that is polyadenylated in human cells. A chimeric gene containing the polyadenylation site from the adenovirus 5 E1A gene is polyadenylated at a site 11 bases downstream from that reported in animal cells. A gene carrying the polyadenylation site from the SV40 early region is polyadenylated some 80 bases upstream from the site that is polyadenylated in animal cells. In all three cases, related mRNAs ending at flanking 'authentic' plant polyadenylation sites can be detected, indicating that the foreign polyadenylation signals are inefficiently utilized in tobacco cells.