Phaseolin Promoter Research Articles

Overexpression of the Phosphatidylcholine:DiacylglycerolCholinephosphotransferase (PDCT) gene increases carbon flux toward triacylglycerol (TAG) synthesis in Camelinasativa seeds

Camelinasativa has considerable promise as a dedicated industrial oilseed crop. Its oil-based blends have been tested and approved as liquid transportation fuels. Previously, we utilized metabolomic and transcriptomic profiling approaches and identified metabolic bottlenecks that control oil production and accumulation in seeds. Accordingly, we selected candidate genes for the metabolic engineering of Camelina. Here we targeted the overexpression of Camelina PDCT gene, which encodes the phosphatidylcholine: diacylglycerol cholinephosphotransferase enzyme. PDCT is proposed as a gatekeeper responsible for the interconversions of diacylglycerol (DAG) and phosphatidylcholine (PC) pools and has the potential to increase the levels of TAG in seeds. To confirm whether increased CsPDCT activity in developing Camelina seeds would enhance carbon flux toward increased levels of TAG and alter oil composition, we overexpressed the CsPDCT gene under the control of the seed-specific phaseolin promoter. Camelina transgenics exhibited significant increases in seed yield (19–56%), seed oil content (9–13%), oil yields per plant (32–76%), and altered polyunsaturated fatty acid (PUFA) content compared to their parental wild-type (WT) plants. Results from [14C] acetate labeling of Camelina developing embryos expressing CsPDCT in culture indicated increased rates of radiolabeled fatty acid incorporation into glycerolipids (up to 64%, 59%, and 43% higher in TAG, DAG, and PC, respectively), relative to WT embryos. We conclude that overexpression of PDCT appears to be a positive strategy to achieve a synergistic effect on the flux through the TAG synthesis pathway, thereby further increasing oil yields in Camelina.

Cystathionine γ-synthase expression in seeds alters metabolic and DNA methylation profiles in Arabidopsis.

Arabidopsis (Arabidopsis thaliana) seeds expressing the feedback-insensitive form of cystathionine γ-synthase (AtD-CGS), the key gene of methionine (Met) synthesis, under the control of a seed-specific phaseolin promoter (SSE plants) show a significant increase in Met content. This elevation is accompanied by increased levels of other amino acids (AAs), sugars, total protein, and starch, which are important from a nutritional aspect. Here, we investigated the mechanism behind this phenomenon. Gas chromatography-mass spectrometry (GC-MS) analysis of SSE leaves, siliques, and seeds collected at 3 different developmental stages showed high levels of Met, AAs, and sugars compared to the control plants. A feeding experiment with isotope-labeled AAs showed an increased flux of AAs from nonseed tissues toward the developing seeds of SSE. Transcriptome analysis of leaves and seeds displayed changes in the status of methylation-related genes in SSE plants that were further validated by methylation-sensitive enzymes and colorimetric assay. These results suggest that SSE leaves have higher DNA methylation rates than control plants. This occurrence apparently led to accelerated senescence, together with enhanced monomer synthesis, which further resulted in increased transport of monomers from the leaves toward the seeds. The developing seeds of SSE plants, however, show reduced Met levels and methylation rates. The results provide insights into the role of Met in DNA methylation and gene expression and how Met affects the metabolic profile of the plant.

The seed-specific transcription factor DPBF2 modulates the fatty acid composition in seeds.

Triacylglycerol (TAG), an ester derived from glycerol and three fatty acids (FAs), is synthesized during seed development and controlled by transcriptional regulation. We examined the mechanism regulating the FA composition of developing Arabidopsis thaliana seeds. The seed‐specific DC3 PROMOTER‐BINDING FACTOR2 (DPBF2) transcription factor was upregulated by LEAFY COTYLEDON2 (LEC2). DPBF2 showed transcriptional activity in yeast and localized to the nucleus in Arabidopsis protoplast cells. The Arabidopsis dpbf2‐1 homozygous T‐DNA mutant and transgenic lines overexpressing of DPBF2 using a seed‐specific phaseolin promoter in wild‐type (WT) Arabidopsis and in dpbf2‐1 showed similar FA composition profiles in their seeds. Their 18:2 and 20:1 FA contents were higher, but 18:1 and 18:3 contents were lower than that of WT. Transcript levels of FATTY ACID DESATURASE2 (FAD2), FAD3, LYSOPHOSPHATIDYLCHOLINE ACYLTRANSFERASE1 (LPCAT1), LPCAT2, PHOSPHATIDYLCHOLINE DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE (PDCT), and FATTY ACID ELONGASE 1 (FAE1) are increased in DPBF2‐overexpressing seeds. Besides, PDCT and FAE1 were upregulated by DPBF2, LEC1‐LIKE (L1L), and NUCLEAR FACTOR‐YC2 (NF‐YC2) transcriptional complex based on tobacco protoplast transcriptional activation assay. These results suggest that DPBF2 effectively modulates the expression of genes encoding FA desaturases, elongase, and acyl‐editing enzymes for modifying the unsaturated FA composition in seeds.

Molecular Pharming of the Recombinant Protein hEGF-hEGF Concatenated with Oleosin Using Transgenic Arabidopsis.

We set out to assess the NIH/3T3 cell proliferation activity of Arabidopsis oil body-expressed recombinant oleosin–hEGF–hEGF protein. Normally, human epidermal growth factor (hEGF) is purified through complex process, however, oleosin fusion technology provides an inexpensive and scalable platform for its purification. Under a phaseolin promoter, we concatenated oleosin gene to double hEGF (hEGF–hEGF) with plant-preferred codons in the expression vectors and the construct was transformed into Arabidopsis thaliana (Arabidopsis). The transgenic Arabidopsis was validated by RT–PCR and the content of recombinant protein oleosin–hEGF–hEGF was quantified by western blot. Subsequently, the proliferation assay and transdermal absorption were determined by MTT method and immunohistochemical staining, respectively. First, the expression level of hEGF was recorded to be 14.83-ng/μL oil body and due to smaller size transgenic oil bodies expressing the recombinant oleosin–hEGF–hEGF, they were more skin permeable than those of control. Second, via the staining intensity of transgenic oil bodies was greater than EGF at all time points via immunohistochemical staining in transdermal absorption process. Lastly, activity assays of oil bodies expressed oleosin–hEGF–hEGF indicated that they stimulated the NIH/3T3 cell proliferation activity. Our results revealed oil-body-expressed oleosin–hEGF–hEGF was potential new material having implications in the field of medicine.

Expression of antigen gp5 of prrs virus in tobaco

PRRS (Porcine reproductive and respiratory syndrome) is the most significant infectious disease currently devastating the swine industry. In order to diversify antigen sources for vaccine production to against PRRSV, co-expressing LTB and PRRSV GP5- an important structure protein GP5 of PRRSV were expressed in tobacco. Genes encoded for GP5 of PRRSV isolated from pigs infected PRRSV in Vietnam in 2010 and was constructed in plant expression vector under the control of seed specific phaseolin promoter and constitutive promoter 35S. The genes were then transferred into the C9-1 tobacco using Agrobacterium. The stable insertion of LTB-GP5genes in genomeoftransgenic tobacco was confirmed by PCR and RT-PCR. Western hybridization indicated that fused protein LTB-GP5 was successful expressed in tobacco seeds. Analysis of transgenic tobaco seeds revealed that under regulation of promoter beta-phaseolin, the level expression of GP5 was significant improved as quantified by ELISA (2.2 % of total soluble protein). This preliminary result is a foundation for expression of GP5 in seeds of other plants in the future.

Expression of a functional recombinant vascular endothelial growth factor 165 (VEGF165) in Arabidopsis thaliana

Abstract Objective Targeting the protein of interest to a particular tissue to achieve high-level expression is an important strategy to increase expression efficiency. The use of the plant seed oil body as a bioreactor can not only increase the amount of target protein, but also reduce the cost of downstream processing. Methods VEGF165 was expressed in Arabidopsis thaliana seeds via oilbody fusion technology. The pKO-VEGF165 vector was construted and transformed into A. thaliana seeds. T3 transgenic seeds was detected by SDS-PAGE and western blot methods. The cell activity was tested by MTT methods. Result The phaseolin promoter was used to drive seed-specific expression of the VEGF 165 gene in transgenic A. thaliana. The coding region of VEGF 165 was fused to the Arabidopsis oleosin sequence to target the protein to the oil bodies in the seeds of transgenic plants. The T-DNA region of recombinant plasmid pKO-VEGF165 was shifted to A. thaliana seeds via the floral-dip method. Protein was analyzed by electrophoresis and protein hybridization analyses. Finally, MTT assays showed that the oleosin-VEGF165 fusion protein played a part in the proliferation of HUVEC cells in vitro. Conclusion Oleosin-VEGF165 was successfully expressed and it had stimulated HUVEC cell proliferation activity.

Expression of biologically recombinant human acidic fibroblast growth factor in Arabidopsis thaliana seeds via oleosin fusion technology

The potential of oleosins to act as carriers for recombinant foreign proteins in plant cells has been established. Using the oleosin fusion technology, the protein can be targeted to oil bodies in oilseeds by fusing it to the N- or C-terminus of oleosin. In this study, aFGF was expressed in Arabidopsis thaliana seeds via oleosin fusion technology. A plant-preferred aFGF gene was synthesized by optimizing codon usage and was fused to the C-terminus of the A. thaliana 18.5kDa oleosin gene. The fusion gene was driven by the phaseolin promoter to confer seed-specific expression of the human acidic fibroblast growth factor in A. thaliana. The T-DNA region of the recombinant plasmid pKO-aFGF was introduced into the genome of Arabidopsis thaliana by the floral dip method. The aFGF protein expression was confirmed by SDS-PAGE and western blotting. The biological activity showed that oil bodies fused to aFGF stimulated NIH/3T3 cell proliferation activity.

Gene Networks and Chromatin and Transcriptional Regulation of the Phaseolin Promoter in Arabidopsis

The complete lack of seed storage protein expression in vegetative tissues and robust expression during embryogenesis makes seed development an ideal system to study tissue-specific expression of genes. The promoter for the Phaseolin (phas) gene, which encodes the major seed storage protein in bean (Phaseolus vulgaris), is activated in two sequential steps: Phaseolus vulgaris ABI3-like factor (Pv-ALF)-dependent potentiation and abscisic acid-mediated activation. In this study, a heterologous in vivo Pv-ALF/phas-GUS (for β-glucuronidase) expression system in transgenic Arabidopsis thaliana leaves was used in conjunction with the powerful RNA-Seq approach to capture transcriptional landscapes of phas promoter expression. Remarkably, expression of over 1300 genes from 11 functional categories coincided with changes in the transcriptional status of the phas promoter. Gene network analysis of induced genes and artificial microRNA-mediated loss-of-function genetic assays identified transcriptional regulators RINGLET 2 (RLT2) and AINTEGUMENTA-LIKE 5 (AIL5) as being essential for phas transcription. Pv-ALF binding to the RLT2 and AIL5 promoter regions was confirmed by electrophoretic mobility shift assay. RLT2 and AIL5 knockdown lines displayed reduced expression of several endogenous seed genes, suggesting that these factors are involved in activation of endogenous Arabidopsis seed storage gene expression. Overall, the identification of these key factors involved in phas activation provides important insight into the two-step transcriptional regulation of seed-specific gene expression.

Transgenic chickpea expressing a recombinant human α1-proteinase inhibitor (α1-PI) driven by a seed-specific promoters from the common bean Phaseolus vulgaris (L.)

The 5′ regulatory sequences of seed-storage protein genes, arcelin 5-I (Arc), phaseolin (Phas) of common bean (Phaseolus vulgaris L.) and legumin (Leg) gene of Cicer arietinum (L.) were evaluated for seed-specific expression of β-glucuronidase (uidA) and recombinant human α1-proteinase inhibitor (α1-PI) in transgenic chickpea. Histochemical assay of transformed plants developed with seed-specific promoters, showed GUS expression in seeds and not in other plant tissues. Fluorometric assay of β-glucuronidase revealed that phaseolin promoter with arcelin 5′ UTR (pPAG) resulted into maximum GUS activity in seeds, followed by somatic tissues and minimal expression in leaves. The expression profile of GUS driven by different seed-specific promoters in chickpea, are in order phaseolin > arcelin > legumin respectively. RT-PCR analysis confirmed that transcripts of β-glucuronidase and α1-PI genes, driven by the phaseolin promoter are stable in chickpea and their accumulation is confined into the seed tissues. Analysis of α1-PI expression in seeds of transgenic chickpea was performed by direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA), residual porcine pancreatic elastase activity assay and Western immunoblotting. Results of DAC-ELISA showed that modified α1-PI gene driven by the phaseolin promoter with arcelin 5′ UTR showed maximum accumulation of α1-PI up to 1.95 μg mg−1 of fresh weight in seeds. Biological activity of recombinant α1-PI, confirmed by elastase inhibition assays exhibiting 0.264 μg mg−1 of active protein. Western immunoblot analysis confirmed the molecular mass of plant-expressed recombinant α1-PI of molecular mass ~50 kDa in the seeds of transgenic chickpea.

Targeted Enhancement of Glutamate-to-γ-Aminobutyrate Conversion in Arabidopsis Seeds Affects Carbon-Nitrogen Balance and Storage Reserves in a Development-Dependent Manner

In seeds, glutamate decarboxylase (GAD) operates at the metabolic nexus between carbon and nitrogen metabolism by catalyzing the unidirectional decarboxylation of glutamate to form γ-aminobutyric acid (GABA). To elucidate the regulatory role of GAD in seed development, we generated Arabidopsis (Arabidopsis thaliana) transgenic plants expressing a truncated GAD from Petunia hybrida missing the carboxyl-terminal regulatory Ca(2+)-calmodulin-binding domain under the transcriptional regulation of the seed maturation-specific phaseolin promoter. Dry seeds of the transgenic plants accumulated considerable amounts of GABA, and during desiccation the content of several amino acids increased, although not glutamate or proline. Dry transgenic seeds had higher protein content than wild-type seeds but lower amounts of the intermediates of glycolysis, glycerol and malate. The total fatty acid content of the transgenic seeds was 50% lower than in the wild type, while acyl-coenzyme A accumulated in the transgenic seeds. Labeling experiments revealed altered levels of respiration in the transgenic seeds, and fractionation studies indicated reduced incorporation of label in the sugar and lipid fractions extracted from transgenic seeds. Comparative transcript profiling of the dry seeds supported the metabolic data. Cellular processes up-regulated at the transcript level included the tricarboxylic acid cycle, fatty acid elongation, the shikimate pathway, tryptophan metabolism, nitrogen-carbon remobilization, and programmed cell death. Genes involved in the regulation of germination were similarly up-regulated. Taken together, these results indicate that the GAD-mediated conversion of glutamate to GABA during seed development plays an important role in balancing carbon and nitrogen metabolism and in storage reserve accumulation.

Changes in Oil Content of Transgenic Soybeans Expressing the Yeast SLC1 Gene

The wild type (Wt) and mutant form of yeast (sphingolipid compensation) genes, SLC1 and SLC1-1, have been shown to have lysophosphatidic acid acyltransferase (LPAT) activities (Nageic et al. in J Biol Chem 269:22156-22163, 1993). Expression of these LPAT genes was reported to increase oil content in transgenic Arabidopsis and Brassica napus. It is of interest to determine if the TAG content increase would also be seen in soybeans. Therefore, the wild type SLC1 was expressed in soybean somatic embryos under the control of seed specific phaseolin promoter. Some transgenic somatic embryos and in both T2 and T3 transgenic seeds showed higher oil contents. Compared to controls, the average increase in triglyceride values went up by 1.5% in transgenic somatic embryos. A maximum of 3.2% increase in seed oil content was observed in a T3 line. Expression of the yeast Wt LPAT gene did not alter the fatty acid composition of the seed oil.

Expression and subcellular targeting of human insulin-like growth factor binding protein-3 in transgenic tobacco plants

Human insulin-like growth factor binding protein-3 (hIGFBP-3) is a multifunctional protein which has high affinity for insulin-like growth factor-I (IGF-I). It combines with IGF-I to form a tertiary complex in circulation, thus regulating the activity of IGF-I. Furthermore, recombinant hIGFBP-3 (rhIGFBP-3) has been found to negatively regulate cell proliferation and induce apoptosis. In this study, we have established an efficient plant bioreactor platform for mass production of rhIGFBP-3. Different expression constructs, driven by the seed-specific phaseolin promoter, were designed and transformed into tobacco plant via Agrobacterium. To enhance protein expression level, the signal peptide (SP) and the C-terminal tetrapeptide AFVY of phaseolin were used to direct rhIGFBP-3 to protein storage vacuole (PSV) in tobacco seed for stable accumulation. Western blot analysis showed that rhIGFBP-3 was successfully synthesized in transgenic tobacco seeds, with the highest protein expression of 800 mug/g dry weight. The localization of rhIGFBP-3 in PSV was also evident by confocal immunofluorescence microscopy. Our results indicated that protein sorting sequences could benefit the expression level of rhIGFBP-3 and it is feasible to use plant as "bio-factory" to produce therapeutic recombinant proteins in large quantity.

Altering Arabidopsis Oilseed Composition by a Combined Antisense‐Hairpin RNAi Gene Suppression Approach

AbstractAntisense (AS) and hairpin (HP) RNA interference (RNAi) targeted gene suppression technologies have been used to modify seed oil composition. Larger numbers of AS transgenics have to be screened to achieve a targeted level of suppression compared to RNAi. We hypothesized combining AS with RNAi might result in enhanced gene suppression compared to either method individually. AS and HP‐RNAi were combined as hairpin antisense (HPAS) constructs containing ~125 bp sense and antisense portions of an untranslated region of the target gene separated by an intron containing an antisense copy of a portion of the target coding region. The Δ12‐desaturase FAD2, the ω3‐desaturase FAD3 and β‐ketoacyl‐ACP synthase (KAS) II were targeted in Arabidopsis to evaluate changes in oil composition with AS, HP and HPAS constructs driven by the phaseolin promoter. Modest but statistically significant enhancements in oilseed phenotypes were observed with HPAS relative to AS and HP‐RNAi. Phenotypes for HPAS suppression of FAD2 and FAD3 were indistinguishable from their strongest mutant alleles. Our data suggest that HPAS may be useful for: (1) achieving levels of suppression comparable to those of gene knockouts in a tissue specific manner. (2) Maximizing suppression of suboptimal RNAi constructs and (3) minimizing the screening of transgenics to achieve desired oilseed composition.

Reduced Oil Accumulation in Cottonseeds Transformed with a Brassica Nonfunctional Allele of a Delta‐12 Fatty Acid Desaturase (FAD2)

In an effort to better understand the mechanisms that regulate oil accumulation and packaging in seeds, transgenic cotton lines were generated using a Brassica napus nonfunctional delta‐12 fatty acid desaturase (FAD2) gene under control of the phaseolin promoter. Seeds of numerous transgenic plant lines had reduced oil content compared with null‐segregating siblings or nontransformed seeds. Seed oil content was quantified by 1H‐NMR, and was reduced to 12% or less of seed weight in transgenics from 20% by weight in nontransformed controls. Light‐ and electron‐microscopic analyses of severely lowered lines, showed a reduction in overall cotyledon thickness and a disruption in cellular and subcellular organization. Lipid bodies and protein bodies were fewer in transgenics, and their size and distribution in cells was different than that in nontransformed seeds. Coincident with reduced storage reserves, sucrose levels were elevated in transgenic seeds. The overall effect of oil suppression was to selectively reduce the size of the embryo, but the seed coat and fiber properties remained unaffected in seeds. In fact there was a significant increase in lint percentage in all oil‐suppressed lines examined. Overall we propose that expression of the nonfunctional Bnfad2 allele in cottonseeds disrupts normal oil biosynthesis and provides for redirection of carbon reserves.

PvALF and FUS3 activate expression from the phaseolin promoter by different mechanisms

Transcription from the phaseolin (phas) promoter requires two major events: chromatin remodeling, mediated by PvALF, a B3 domain factor, and activation by an ABA-induced signal transduction cascade. Expression from phas is normally seed-specific, but high levels of expression in leaves can be obtained by ectopic expression of PvALF. Here, the system was used to compare the ability of PvALF and Arabidopsis FUS3, another B3 domain transcription factor that lacks the N-terminal activation and B1 domain present in PvALF, to activate phas expression in vegetative tissues. When compared to PvALF-mediated phas activation in the presence of ABA, a delay in phas activation was observed in the presence of both FUS3 and ABA in vegetative tissue. Significant differences in histone modifications at the phas promoter were mediated by FUS3 and PvALF, suggesting that they function through different epigenetic mechanisms. The relationship between PvALF and ABI5, a bZIP transcription factor, in mediating phas expression was also evaluated. Interestingly, over-expression of ABI5 rendered phas expression ABA-independent in the presence of PvALF. Changes in phas activity in different regions within seed embryos were demonstrated using abi5 mutants. Our results show that (1) redundant factors, such as PvALF and FUS3, employ different mechanisms to regulate their common target gene (phas); (2) ABI5, and possibly other redundant bZIP factors, act downstream of ABA in modulating phas expression in the presence of PvALF.

Ordered Histone Modifications Are Associated with Transcriptional Poising and Activation of the phaseolin Promoter

The phaseolin (phas) promoter drives copious production of transcripts encoding the protein phaseolin during seed embryogenesis but is silent in vegetative tissues, in which a nucleosome is positioned over its three-phased TATA boxes. Transition from the inactive state in transgenic Arabidopsis thaliana leaves was accomplished by ectopic expression of the transcription factor Phaseolus vulgaris ABI3-like factor (ALF) and application of abscisic acid (ABA). Placement of hemagglutinin-tagged ALF expression under the control of an estradiol-inducible promoter permitted chromatin immunoprecipitation analysis of chronological changes in histone modifications, notably increased acetylation of H3-K9 and H4-K12, as phas chromatin was remodeled (potentiated). A different array of changes, including acetylation of H3-K14 and methylation of H3-K4, was found to be associated with ABA-mediated activation. Thus, temporal separation of phas potentiation from activation revealed that histone H3 and H4 Lys residues are not globally hyperacetylated during phas expression. Whereas decreases in histone H3 and H4 levels were detected during ALF-mediated remodeling, slight increases occurred after ABA-mediated activation, suggesting the restoration of histone-phas interactions or the replacement of histones in the phas chromatin. The observed histone modifications provide insight into factors involved in the euchromatinization and activation of a plant gene and expand the evidence for histone code conservation among eukaryotes.

Agrobacterium-mediated transformation of faba bean (Vicia faba L.) using embryo axes

A system for the production of transgenic faba bean by Agrobacterium-mediated transformation was developed. This system is based upon direct shoot organogenesis after transformation of meristematic cells derived from embryo axes. Explants were co-cultivated with A. tumefaciens strain EHA105/pGlsfa, which harbored a binary vector containing a gene encoding a sulphur rich sunflower albumin (SFA8) linked to the bar gene. Strain EHA 101/pAN109 carrying the binary plasmid containing the coding sequence of a mutant aspartate kinase gene (lysC) from E. coli in combination with neomycinphosphotransferase II gene (nptII) was used as well. The coding sequences of SFA8 and LysC genes were fused to seed specific promoters, either Vicia faba legumin B4 promoter (LeB4) or phaseolin promoter, respectively. Seven phosphinothricin (PPT) resistant clones from Mythos and Albatross cultivars were recovered. Integration, inheritance and expression of the transgenes were confirmed by Southern blot, PCR, enzyme activity assay and Western blot.

Expression of a Stokesia laevis epoxygenase gene

Epoxy fatty acids have a number of important uses and there is interest in enzymes catalyzing their synthesis from renewable sources. Both cytochrome P450 monooxygenases and divergent forms of di-iron desaturases are known to produce epoxy fatty acids in plants. Degenerate primers based on conserved sequences of Δ 12 desaturase-like genes led to the isolation of an epoxygenase gene from Stokesia laevis. The cDNA is 1.4 kb and it encodes 378 amino acids. The similarities of this gene at the amino acid sequence level with epoxygenases of Vernonia and Crepis, and the Δ 12 desaturases of soybean, FAD2-1 and FAD2-2, are 84%, 69%, 49%, and 55%, respectively. When the vector, pYES2, was used to transform yeast, epoxy fatty acid formation was observed in the cells. The effects of electron donors in the yeast expression system were tested but cytochrome b 5 and cytochrome b 5 reductase genes from Arabidopsis thaliana co-expressed with the epoxygenase had little effect on vernolic acid accumulation in the yeast. Finally, this gene, driven by a seed-specific phaseolin promoter, was cloned into a TDNA-vector and transferred into Arabidopsis plants. The results showed that T 2 seeds of transgenic Arabidopsis expressing the Stokesia gene accumulated vernolic acid but no vernolic acid was detected in control plants. Northern blot analysis indicates this S. laevis epoxygenase gene is expressed mainly in developing seeds and no transcript was detected in leaves or roots.

Control of seed germination in transgenic plants based on the segregation of a two-component genetic system.

We have developed a repressible seed-lethal (SL) system aimed at reducing the probability of transgene introgression into a population of sexually compatible plants. To evaluate the potential of this method, tobacco plants were transformed with an SL construct comprising gene 1 and gene 2 from Agrobacterium tumefaciens whereby gene 1 was controlled by the seed-specific phaseolin promoter modified to contain a binding site for the Escherichia coli TET repressor (R). The expression of this construct allows normal plant and seed development but inhibits seed germination. Plants containing the SL construct were crossed with plants containing the tet R gene to derive plant lines where the expression of the SL construct is repressed. Plant lines that contained both constructs allowed normal seed formation and germination, whereas seeds in which the SL construct was separated from the R gene through segregation did not germinate. The requirements of such a method to efficiently control the flow of novel traits among sexually compatible plants are discussed.

Cre recombinase expression can result in phenotypic aberrations in plants.

The cre recombinase gene was stably introduced and expressed in tomato, petunia and Nicotiana tabacum. Some plants expressing the cre gene driven by a CaMV 35S promoter displayed growth retardation and a distinct pattern of chlorosis in their leaves. Although no direct relation can be proven between the phenotype and cre expression, aberrant phenotypes always co-segregate with the transgene, which strongly suggests a correlation. The severity of the phenotype does not correlate with the level of steady-state mRNA in mature leaves, but with the timing of cre expression during organogenesis. The early onset of cre expression in tomato is correlated with a more severe phenotype and with higher germinal transmission frequencies of site-specific deletions. No aberrant phenotype was observed when a tissue-specific phaseolin promoter was used to drive the cre gene. The data suggest that for the application of recombinases in plants, expression is best limited to specific tissues and a short time frame.