Production of the isoflavones genistein and daidzein in non-legume dicot and monocot tissues.

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.

Seed-specific gene activation mediated by the Cre/lox site-specific recombination system.

The Cre/lox site-specific recombination system was used to activate a transgene in a tissue-specific manner. Cre-mediated activation of a beta-glucuronidase marker gene, by removal of a lox-bounded blocking fragment, allowed the visualization of the activation process. By using seed-specific promoters, the timing and efficiency of gene activation could be followed within the developing tobacco (Nicotiana tabacum) embryo. To serve as a basis for analyzing gene expression after-Cre-mediated activation, the timing and patterns of expression of the promoters of the genes encoding French bean (Phaseolus vulgaris) beta-phaseolin and the alpha' subunit of soybean (Glycine max) beta-conglycinin, as well as the cauliflower mosaic virus 35S promoter, were studied in developing transgenic tobacco embryos using the same visual marker. These seed-specific promoters were expressed earlier than anticipated. The 35S promoter was expressed earlier than the seed-specific promoters, but not in globular-stage embryos. Cre-mediated gene activation occurred approximately 1 d after promoter activation, based on developmental staging, and spread progressively throughout the embryo. The timing of gene activation was varied by altering Cre expression. Efficient Cre expression ultimately directed gene activation throughout the model tissue, whereas inefficient Cre expression resulted in mosaic tissue. Limited gene activation provides a system for cell lineage and developmental analyses.

Plant Expression of a Bacterial Cytochrome P450 That Catalyzes Activation of a Sulfonylurea Pro-Herbicide.

The Streptomyces griseolus gene encoding herbicide-metabolizing cytochrome P450SU1 (CYP105A1) was expressed in transgenic tobacco (Nicotiana tabacum). Because this P450 can be reduced by plant chloroplast ferredoxin in vitro, chloroplast-targeted and nontargeted expression were compared. Whereas P450SU1 antigen was found in the transgenic plants regardless of the targeting, only those with chloroplast-directed enzyme performed P450SU1-mediated N-dealkylation of the sulfonylurea 2-methylethyl-2,3-dihydro-N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]-1, 2-benzoisothiazole- 7-sulfonamide-1,1-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 P450SU1 are susceptible to injury from R7402 treatment that is harmless to plants without P450SU1. Thus, P450SU1 expression and R7402 treatment can be used as a negative selection system in plants. Furthermore, expression of P450SU1 from a tissue-specific promoter can sequester production of the phytotoxic R7402 metabolite to a single plant tissue. In tobacco expressing P450SU1 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.

Directed excision of a transgene from the plant genome.

The effectiveness of loxP-Cre directed excision of a transgene was examined using phenotypic and molecular analyses. Two methods of combining the elements of this system, re-transformation and cross pollination, were found to produce different degrees of excision in the resulting plants. Two linked traits, beta-glucuronidase (GUS) and a gene encoding sulfonylurea-resistant acetolactate synthase (ALSr), were integrated into the genome of tobacco and Arabidopsis. The ALSr gene, bounded by loxP sites, was used as the selectable marker for transformation. The directed loss of the ALSr gene through Cre-mediated excision was demonstrated by the loss of resistance to sulfonylurea herbicides and by Southern blot analysis. The beta-glucuronidase gene remained active. The excision efficiency varied in F1 progeny of different lox and Cre parents and was correlated with the Cre parent. Many of the lox x Cre F1 progeny were chimeric and some F2 progeny retained resistance to sulfonylureas. Re-transformation of lox/ALS/lox/GUS tobacco plants with cre led to much higher efficiency of excision. Lines of tobacco transformants carrying the GUS gene but producing only sulfonylurea-sensitive progeny were obtained using both approaches for introducing cre. Similarly, Arabidopsis lines with GUS activity but no sulfonylurea resistance were generated using cross pollinations.

Comparison of increased expression of wild-type and herbicide-resistant acetolactate synthase genes in transgenic plants, and indication of posttranscriptional limitation on enzyme activity.

Genes encoding wild type acetolactate synthase (ALS) and a sulfonylurea herbicide-resistant form of the enzyme, isolated from Arabidopsis thaliana, were expressed in transgenic Nicotiana tabacum plants under the control of their native promoters or of the highly active cauliflower mosaic virus 35S promoter. Expression of the wild type coding region from the 35S promoter resulted in a small, threefold increase in sulfonylurea tolerance above the levels measured in tissue expressing the native wild type gene. A much larger, 300-fold increase in herbicide tolerance was conferred by the mutant gene encoding a herbicide-resistant ALS. An additional 10-fold increase in tolerance was attained by expressing this coding region from the 35S promoter. The increase in both wild type and mutant gene expression directed by the 35S promoter resulted in over 25-fold higher levels of ALS messenger RNA in some transformants as compared with those expressing the native genes. However, ALS specific activity increased at most twofold, indicating that the amount of functional enzyme and messenger RNA are not correlated.

Properties of an isolated transcription stimulating sequence derived from the cauliflower mosaic virus 35S promoter.

As a highly active plant viral promoter that is able to function in a wide variety of cell types, the cauliflower mosaic virus (CaMV) 35S promoter has the potential for harboring a plant enhancer element. We tested this possibility and demonstrated that a 338 base pair fragment isolated from the region upstream of the 35S TATA box can increase the expression of a low-activity heterologous promoter up to the level observed for the intact 35S promoter. This fragment is fully active in both orientations when placed 150 base pairs upstream of the transcription start site. However, the activity of this fragment is sensitive to location, demonstrating a reduction in activity and loss of orientation-independent function when the distance from the transcription start site is increased. By assaying fragments of different sizes, we have also characterized regions that are functional in directing the stimulation of the heterologous promoter.

Soybean protoplast culture and direct gene uptake and expression by cultured soybean protoplasts.

A method was developed for culturing protoplasts freshly isolated from developing soybean (Glycine max L.) cotyledons. First cell divisions were observed within 5 days after protoplast isolation and microcalli, consisting of about 20 cells, were formed within 10 days. Thirty days after protoplast isolation, callus tissues were observed without the aid of a microscope. A 30 to 50% plating efficiency was consistently obtained. Using a polyethylene glycol-electroporation technique, DNA was introduced into these protoplasts. The protoplasts were then cultured to form callus. Chloramphenicol acetyltransferase (CAT) activity was detected in protoplast cultures 6 hours after introduction of a 35S-CAT-nopaline synthase 3' chimeric gene. The highest CAT activity was detected in 3-day-old electroporated protoplast cultures, indicating transient expression of the introduced gene. Some CAT activity was detected in 40-day-old callus cultures and in geneticin (G418) selected callus tissues which also received a chimeric neomycin phosphotransferase II gene, indicating the presence of stable transformants. A control chimeric gene with an inverted 35S promoter failed to produce any CAT activity in this system.

Plant cells do not properly recognize animal gene polyadenylation signals.

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.

Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter.

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.

Structure and 5'-termini of the large and 19 S RNA transcripts encoded by the cauliflower mosaic virus genome.

Cauliflower mosaic virus-infected leaves accumulate two major RNA species late in infection-a 19 S RNA and a large RNA transcript. We have found that the large transcript is approximately full genome length. The 3'-end of the large transcript is located at or near a site corresponding to the 3'-end of the 19 S RNA. The 5'-end of the large transcript maps near its own 3'-end and only about 100 bp downstream from the terminator codon for coding region VI. The 5'-end of the large transcript is situated at the beginning of the large intergenic region, about 600 bp upstream from the single-strand break in the transcribed viral DNA strand. The location of this site is of particular interest because it suggests that synthesis of the large transcript proceeds across the DNA break site. The 5'-end of the 19 S RNA maps in the small intergenic region between coding regions V and VI about 20-30 bp upstream from the initiation codon for coding region VI. By comparing the transcription initiation sites to DNA sequences for the CaMV genome, it can be shown that TATATAA sequences lie 20-30 bp upstream from the 5'-ends of both the 19 S and large transcripts.

Structure of the 19 S RNA transcript encoded by the cauliflower mosaic virus genome.

Turnip leaves infected with cauliflower mosaic virus accumulate several virus-derived RNA species. These include a large transcript (>25 S) and a 19 S transcript. R-looping and diazo-cellulose filter hybridization techniques show that the 19 S RNA is derived from a contiguous segment of the CaMV genome which includes EcoR1-b and -d DNA fragments. This region encodes the synthesis of a nonvirion protein called P66. The 3' end of the 19 S transcript is polyadenylated and terminates near the single strand break in the alpha-strand of the genome. The 5' end of the transcript starts in or near the EcoR1-d fragment, near position zero on the map described by Hull and Howell (1978).

The identification, mapping, and characterization of mRNA for P66, a cauliflower mosaic virus-coded protein.

Turnip leaves infected with cauliflower mosaic virus accumulate variable amounts of a nonvirion protein of 66,000 MW. Tryptic fingerprint analysis showed that this protein is probably not related to the major virus coat protein (37,000 MW). RNA extracted from infected leaves directs the synthesis of a 66,000 MW protein, P66, in a wheat germ protein-synthesizing system. P66 synthesis in vitro could be arrested by prior hybridization of infected leaf RNA to viral DNA restriction fragments indicating that P66 is encoded by the virus genome and specifically by EcoRI fragment b. RNA coding for P66 sediments at about 18 S, binds, in part, to oligo dT-cellulose, and is sensitive to 7-methyl-GTP inhibition.