Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility.

BACKGROUND: Alfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production. RESULTS: In this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line. CONCLUSION: Modifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines.

Comparability of imazapyr-resistant Arabidopsis created by transgenesis and mutagenesis.

The Arabidopsis CSR1 gene codes for the enzyme acetohydroxyacid synthase (AHAS, EC 2.2.1.6), also known as acetolactate synthase, which catalyzes the first step in branched-chain amino acid biosynthesis. It is inhibited by several classes of herbicides, including the imidazolinone herbicides, such as imazapyr; however, a substitution mutation in csr1-2 (Ser-653-Asn) confers selective resistance to the imidazolinones. The transcriptome of csr1-2 seedlings grown in the presence of imazapyr has been shown in a previous study (Manabe in Plant Cell Physiol 48:1340-1358, 2007) to be identical to that of wild-type seedlings indicating that AHAS is the sole target of imazapyr and that the mutation is not associated with pleiotropic effects detectable by transcriptome analysis. In this study, a lethal null mutant, csr1-7, created by a T-DNA insertion into the CSR1 gene was complemented with a randomly-inserted 35S/CSR1-2/NOS transgene in a subsequent genetic transformation event. A comparison of the csr1-2 substitution mutant with the transgenic lines revealed that all were resistant to imazapyr; however, the transgenic lines yielded significantly higher levels of resistance and greater biomass accumulation in the presence of imazapyr. Microarray analysis revealed few differences in their transcriptomes. The most notable was a sevenfold to tenfold elevation in the CSR1-2 transcript level. The data indicate that transgenesis did not create significant unintended pleiotropic effects on gene expression and that the mutant and transgenic lines were highly similar, except for the level of herbicide resistance.

Identification of two anthocyanidin reductase genes and three red-brown soybean accessions with reduced anthocyanidin reductase 1 mRNA, activity, and seed coat proanthocyanidin amounts.

Anthocyanidin reductase (ANR; EC 1.3.1.77) catalyzes a key step in the biosynthesis of proanthocyanidins (PAs; also known as condensed tannins), flavonoid metabolites responsible for the brown pigmentation of seeds. Here, two ANR genes (ANR1 and ANR2) from the seed coat of brown soybean (Glycine max (L.) Merr.) have been isolated and their enzymatic function confirmed for the reduction of cyanidin to (-)-epicatechin in vitro. Biochemical and genetic comparisons of soybean lines differing in seed coat color revealed three red-brown lines to exhibit major reductions in the amounts of soluble PAs in the seed coat compared to brown soybean lines. Two spontaneous mutants with red-brown grain color had reduced ANR1 gene expression in the seed coat, and an EMS-mutagenized red-brown mutant had nonsynonymous substitutions that resulted in slightly reduced ANR1 activity in vitro. These results suggest that defects in the ANR1 gene can be associated with red-brown soybean grain color. These results suggest that suppressing ANR1 gene expression or activity may be a rational approach toward engineering seed coat color to enable the visual identification of genetically modified soybean grains.

Coloring genetically modified soybean grains with anthocyanins by suppression of the proanthocyanidin genes ANR1 and ANR2.

Detection and quantification of the levels of adventitious presence of genetically modified (GM) soybeans in non-GM grain shipments currently requires sophisticated tests that can have issues with their reproducibility. We show here that pigment biosynthesis in the soybean seed coat can be manipulated to provide a distinct color that would enable the simple visible detection of the GM soybean grain. We observed that a distinct red-brown grain color could be engineered by the simultaneous suppression of two proanthocyanidin (PA) genes, ANTHOCYANIDIN REDUCTASE1 (ANR1) and ANR2. Multiple reaction monitoring by liquid chromatography tandem mass spectrometry was used to quantify differentially accumulated seed coat metabolites, and revealed the redirection of metabolic flux into the anthocyanin pigment pathway and unexpectedly the flavonol-3-O-glucoside pathway. The upregulations of anthocyanin isogenes (DFR1 and GST26) and the anthocyanin/flavonol-3-O-glycosyltransferase (UGT78K2) were identified by quantitative RT-PCR to be endogenous feedback and feedforward responses to overaccumulation of upstream flavonoid intermediates resulting from ANR1 and ANR2 suppressions. These results suggested the transcription of flavonoid genes to be a key component of the mechanism responsible for the redirection of metabolite flux. This report identifies the suppression of PA genes to be a novel approach for engineering pigmentation in soybean grains.

Combined analysis of transcriptome and metabolite data reveals extensive differences between black and brown nearly-isogenic soybean (Glycine max) seed coats enabling the identification of pigment isogenes.

BACKGROUND: The R locus controls the color of pigmented soybean (Glycine max) seeds. However information about its control over seed coat biochemistry and gene expressions remains limited. The seed coats of nearly-isogenic black (iRT) and brown (irT) soybean (Glycine max) were known to differ by the presence or absence of anthocyanins, respectively, with genes for only a single enzyme (anthocyanidin synthase) found to be differentially expressed between isolines. We recently identified and characterized a UDP-glycose:flavonoid-3-O-glycosyltransferase (UGT78K1) from the seed coat of black (iRT) soybean with the aim to engineer seed coat color by suppression of an anthocyanin-specific gene. However, it remained to be investigated whether UGT78K1 was overexpressed with anthocyanin biosynthesis in the black (iRT) seed coat compared to the nearly-isogenic brown (irT) tissue.In this study, we performed a combined analysis of transcriptome and metabolite data to elucidate the control of the R locus over seed coat biochemistry and to identify pigment biosynthesis genes. Two differentially expressed late-stage anthocyanin biosynthesis isogenes were further characterized, as they may serve as useful targets for the manipulation of soybean grain color while minimizing the potential for unintended effects on the plant system. RESULTS: Metabolite composition differences were found to not be limited to anthocyanins, with specific proanthocyanidins, isoflavones, and phenylpropanoids present exclusively in the black (iRT) or the brown (irT) seed coat. A global analysis of gene expressions identified UGT78K1 and 19 other anthocyanin, (iso)flavonoid, and phenylpropanoid isogenes to be differentially expressed between isolines. A combined analysis of metabolite and gene expression data enabled the assignment of putative functions to biosynthesis and transport isogenes. The recombinant enzymes of two genes were validated to catalyze late-stage steps in anthocyanin biosynthesis in vitro and expression profiles of the corresponding genes were shown to parallel anthocyanin biosynthesis during black (iRT) seed coat development. CONCLUSION: Metabolite composition and gene expression differences between black (iRT) and brown (irT) seed coats are far more extensive than previously thought. Putative anthocyanin, proanthocyanidin, (iso)flavonoid, and phenylpropanoid isogenes were differentially-expressed between black (iRT) and brown (irT) seed coats, and UGT78K2 and OMT5 were validated to code UDP-glycose:flavonoid-3-O-glycosyltransferase and anthocyanin 3'-O-methyltransferase proteins in vitro, respectively. Duplicate gene copies for several enzymes were overexpressed in the black (iRT) seed coat suggesting more than one isogene may have to be silenced to engineer seed coat color using RNA interference.

Role of HD2 genes in seed germination and early seedling growth in Arabidopsis.

The Arabidopsis HD2 family of histone deacetylases consist of 4 members (HD2A, HD2B, HD2C, HD2D) that play diverse roles in plant development and physiology through chromatin remodelling. Here, we show that the transcripts of HD2 family members selectively accumulate in response to glucose through a HXK1-independent signal transduction pathway during the early stages of seedling growth. Germination was enhanced in hd2a null mutants relative to wild-type seeds. In contrast, hd2c mutants were restrained in germination relative to wild-type seeds. In hd2a/hd2c double mutants, germination was restored to wild-type levels. The data suggests that HD2A and HD2C may have different and opposing functions in germination with the glucose/HD2A pathway acting to restrain germination and the HD2C pathway acting to enhance germination. These pathways may function early in the regulation of seedling germination, independently of the glucose/HXK1/ABA signal transduction pathway, to fine tune the onset of germination.

Control of somatic embryogenesis and embryo development by AP2 transcription factors.

Members of the AP2 family of transcription factors, such as BABY BOOM (BBM), play important roles in cell proliferation and embryogenesis in Arabidopsis thaliana (AtBBM) and Brassica napus (BnBBM) but how this occurs is not understood. We have isolated three AP2 genes (GmBBM1, GmAIL5, GmPLT2) from somatic embryo cultures of soybean, Glycine max (L.) Merr, and discovered GmBBM1 to be homologous to AtBBM and BnBBM. GmAIL5 and GmPLT2 were homologous to Arabidopsis AINTEGUMENTA-like5 (AIL5) and PLETHORA2 (PLT2), respectively. Constitutive expression of GmBBM1 in Arabidopsis induced somatic embryos on vegetative organs and other pleiotropic effects on post-germinative vegetative organ development. Sequence comparisons of BBM orthologues revealed the presence of ten sequence motifs outside of the AP2 DNA-binding domains. One of the motifs, bbm-1, was specific to the BBM-like genes. Deletion and domain swap analyses revealed that bbm-1 was important for somatic embryogenesis and acted cooperatively with at least one other motif, euANT2, in the regulation of somatic embryogenesis and embryo development in transgenic Arabidopsis. The results provide new insights into the mechanisms by which BBM governs embryogenesis.

Functional characterization of a UDP-glucose:flavonoid 3-O-glucosyltransferase from the seed coat of black soybean (Glycine max (L.) Merr.).

The seed coats of black soybean (Glycine max (L.) Merr.) accumulate red (cyanidin-), blue (delphinidin-), purple (petunidin-), and orange (pelargonidin-based) anthocyanins almost exclusively as 3-O-glucosides; however, the responsible enzyme has not been identified. In this study, the full-length cDNA which encodes the enzyme that catalyzes the final step in anthocyanin biosynthesis, namely UDP-glucose:flavonoid 3-O-glucosyltransferase (UGT78K1), was isolated from the seed coat tissue of black soybean using rapid amplification of cDNA ends (RACE). Of the 28 flavonoid substrates tested, the purified recombinant protein glucosylated only anthocyanidins and flavonols, and demonstrated strict 3-OH regiospecificity. Galactose could also be transferred with relatively low activity to the 3-position of cyanidin or delphinidin in vitro. These findings are consistent with previous reports of mainly 3-O-glucosylated and minor amounts of 3-O-galactosylated anthocyanins in the seed coat of black soybean. The recombinant enzyme exhibited pronounced substrate inhibition by cyanidin at 100 microM acceptor concentration. Transfer of UGT78K1 into the Arabidopsis T-DNA mutant (ugt78d2) deficient in anthocyanidin and flavonol 3-O-glucosyltransferase activity, restored the accumulation of anthocyanins and flavonols, suggesting the in vivo function of the enzyme as a flavonoid 3-O-glucosyltransferase. Genomic and phylogenetic analyses suggest the existence of three additional soybean sequences with high similarity to UGT78K1. RT-PCR confirmed the co-expression of one of these genes (Glyma08g07130) with UGT78K1 in the seed coat of black soybean, suggesting possible functional redundancies in anthocyanin biosynthesis in this tissue.

Soybean peroxidase propeptides are functional signal peptides and increase the yield of a foreign protein.

Elements that contribute to the high, stable yield of soybean peroxidase (SBP) in soybean seed coats can be exploited in the development of this tissue as a protein production platform. SBP contains an N-terminal and a C-terminal propeptide that are predicted to direct vacuolar targeting; this may be one factor that contributes to its high yield and stability. We characterized the function of the SBP propeptides and investigated their ability to increase the yield of a foreign protein in a heterologous plant system. SBP propeptides are functional signal peptides capable of directing vacuolar transport in Arabidopsis. The use of these propeptides as well as an endoplasmic reticulum (ER)-retention signal to direct a foreign protein to the apoplast, ER, or vacuole can significantly increase yield and will therefore be useful for the development of the seed coat as a protein production platform. We also demonstrate that growth conditions may have a significant impact on the yield of a foreign protein and that this may be subcellular compartment-specific.

Transcriptome analysis reveals absence of unintended effects in drought-tolerant transgenic plants overexpressing the transcription factor ABF3.

BACKGROUND: Plants engineered for abiotic stress tolerance may soon be commercialized. The engineering of these plants typically involves the manipulation of complex multigene networks and may therefore have a greater potential to introduce pleiotropic effects than the simple monogenic traits that currently dominate the plant biotechnology market. While research on unintended effects in transgenic plant systems has been instrumental in demonstrating the substantial equivalence of many transgenic plant systems, it is essential that such analyses be extended to transgenic plants engineered for stress tolerance. Drought-tolerant Arabidopsis thaliana were engineered through overexpression of the transcription factor ABF3 in order to investigate unintended pleiotropic effects. In order to eliminate position effects, the Cre/lox recombination system was used to create control plant lines that contain identical T-DNA insertion sites but with the ABF3 transgene excised. This additionally allowed us to determine if Cre recombinase can cause unintended effects that impact the transcriptome. RESULTS: Microarray analysis of control plant lines that underwent Cre-mediated excision of the ABF3 transgene revealed only two genes that were differentially expressed in more than one plant line, suggesting that the impact of Cre recombinase on the transcriptome was minimal. In the absence of drought stress, overexpression of ABF3 had no effect on the transcriptome, but following drought stress, differences were observed in the gene expression patterns of plants overexpressing ABF3 relative to control plants. Examination of the functional distribution of the differentially expressed genes revealed strong similarity indicating that unintended pathways were not activated. CONCLUSIONS: The action of ABF3 is tightly controlled in Arabidopsis. In the absence of drought stress, ectopic activation of drought response pathways does not occur. In response to drought stress, overexpression of ABF3 results in a reprogramming of the drought response, which is characterized by changes in the timing or strength of expression of some drought response genes, without activating any unexpected gene networks. These results illustrate that important gene networks are highly regulated in Arabidopsis and that engineering stress tolerance may not necessarily cause extensive changes to the transcriptome.

Strategies to mitigate transgene-promoter interactions.

The expression pattern of tissue-specific promoters in transgenes can be influenced by promoter/enhancer elements employed for the expression of selectable marker genes or elements found in DNA flanking the insertion site. We have developed an analytical system in Arabidopsis thaliana to investigate strategies useful in blocking or reducing nonspecific interactions. These experiments confirm that the DNA configuration and the insertion of spacer DNA aid in the appropriate expression of tissue-specific promoters. It is also demonstrated that the novel tobacco cryptic promoter (tCUP), when used to replace the cauliflower mosaic virus (CaMV) 35S promoter/enhancer, does not show nonspecific interactions. Furthermore, it is shown that insulators isolated from yeast and animals may have potential application in plants. Our results may allow the design of strategies that, individually or in combination, can be used to minimize nonspecific interactions and to design vectors for individual tissue-specific promoters.

Selectable marker genes and unintended changes to the plant transcriptome.

The intended effect of a selectable marker gene is to confer a novel trait that allows for the selection and recovery of transgenic plants. Unintended effects may also occur as a result of interactions between the selectable marker gene or its regulatory elements and genetic elements at the site of insertion. These are called position effects. Other unintended effects may occur if the selectable marker gene has a range of pleiotropic effects related to the functional and regulatory domains within the coding region or the regulatory elements used to drive expression. Both pleiotropic and position effects may generate unpredictable events depending on the process used for transgenesis and the state of knowledge associated with the selectable marker gene. Although some selectable marker genes, such as the neomycin phosphotransferase type II gene (nptII), have no pleiotropic effects on the transcriptomes of transgenic plants, others, such as the bialaphos resistance gene (bar), have pleiotropic effects. These must be clearly understood and accounted for when evaluating the expression patterns conferred by other co-transforming transgenes under study. The number and kinds of selectable marker genes are large. A detailed understanding of their unintended effects is needed to develop transgenic strategies that will minimize or eliminate unintended and unpredictable changes to plants with newly inserted genes.

The pleiotropic effects of the bar gene and glufosinate on the Arabidopsis transcriptome.

The Arabidopsis transcriptome was studied using the Affymetrix Arabidopsis ATH1 GeneChip in wild-type plants and glufosinate-tolerant transgenic plants expressing the bialaphos resistance (bar) gene. Pleiotropic effects were specifically generated in the transcriptomes of transgenic plants by both the bar gene and glufosinate treatments. In the absence of glufosinate, four genes were differentially expressed in the transgenic lines and another 80 genes were differentially expressed in the presence of glufosinate, 29 of which were specific to transgenic plants. In contrast, the number of differentially expressed genes specific to wild-type plants was 194 during the early response at 6 h of glufosinate treatment, and increased to 3711 during the late response at 48 h. Although the wild-type plants undergo extensive transcriptional reprofiling in response to herbicide-induced stress and, finally, plant death, the transgenic plants appear to activate other detoxification processes to offset the toxic effects of the residual herbicide or its derivatives. This study provides the first description of the pleiotropic effects of the bar gene and glufosinate on the plant transcriptome.

CSR1, the sole target of imidazolinone herbicide in Arabidopsis thaliana.

The imidazolinone-tolerant mutant of Arabidopsis thaliana, csr1-2(D), carries a mutation equivalent to that found in commercially available Clearfield crops. Despite their widespread usage, the mechanism by which Clearfield crops gain imidazolinone herbicide tolerance has not yet been fully characterized. Transcription profiling of imazapyr (an imidazolinone herbicide)-treated wild-type and csr1-2(D) mutant plants using Affymetrix ATH1 GeneChip microarrays was performed to elucidate further the biochemical and genetic mechanisms of imidazolinone resistance. In wild-type shoots, the genes which responded earliest to imazapyr treatment were detoxification-related genes which have also been shown to be induced by other abiotic stresses. Early-response genes included steroid sulfotransferase (ST) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), as well as members of the glycosyltransferase, glutathione transferase (GST), cytochrome P450, ATP-binding cassette (ABC) transporter, multidrug and toxin extrusion (MATE) and alternative oxidase (AOX) protein families. Later stages of the imazapyr response involved regulation of genes participating in biosynthesis of amino acids, secondary metabolites and tRNA. In contrast to the dynamic changes in the transcriptome profile observed in imazapyr-treated wild-type plants, the transcriptome of csr1-2(D) did not exhibit significant changes following imazapyr treatment, compared with mock-treated csr1-2(D). Further, no substantial difference was observed between wild-type and csr1-2(D) transcriptomes in the absence of imazapyr treatment. These results indicate that CSR1 is the sole target of imidazolinone and that the csr1-2(D) mutation has little or no detrimental effect on whole-plant fitness.

Combined transcriptome and proteome analysis identifies pathways and markers associated with the establishment of rapeseed microspore-derived embryo development.

Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants.

pORE: a modular binary vector series suited for both monocot and dicot plant transformation.

We present a series of 14 binary vectors suitable for Agrobacterium-mediated transformation of dicotyledonous plants and adaptable for biolistic transformation of monocotyledonous plants. The vector size has been minimized by eliminating all non-essential elements from the vector backbone and T-DNA regions while maintaining the ability to replicate independently. The smallest of the vector series is 6.3 kb and possesses an extensive multiple cloning site with 21 unique restriction endonuclease sites that are compatible with common cloning, protein expression, yeast two-hybrid and other binary vectors. The T-DNA region was engineered using a synthetic designer oligonucleotide resulting in an entirely modular system whereby any vector element can be independently exchanged. The high copy number ColE1 origin of replication has been included to enhance plasmid yield in Escherichia coli. FRT recombination sites flank the selectable marker cassette regions and allow for in planta excision by FLP recombinase. The pORE series consists of three basic types; an 'open' set for general plant transformation, a 'reporter' set for promoter analysis and an 'expression' set for constitutive expression of transgenes. The sets comprise various combinations of promoters (P (HPL), P (ENTCUP2) and P (TAPADH)), selectable markers (nptII and pat) and reporter genes (gusA and smgfp).

HISTONE DEACETYLASE19 is involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis.

Histone acetylation is modulated through the action of histone acetyltransferases and deacetylases, which play key roles in the regulation of eukaryotic gene expression. Previously, we have identified a yeast histone deacetylase REDUCED POTASSIUM DEPENDENCY3 (RPD3) homolog, HISTONE DEACETYLASE19 (HDA19) (AtRPD3A), in Arabidopsis thaliana. Here, we report further study of the expression and function of HDA19. Analysis of Arabidopsis plants containing the HDA19:beta-glucuronidase fusion gene revealed that HDA19 was expressed throughout the life of the plant and in most plant organs examined. In addition, the expression of HDA19 was induced by wounding, the pathogen Alternaria brassicicola, and the plant hormones jasmonic acid and ethylene. Using green fluorescent protein fusion, we demonstrated that HDA19 accumulated in the nuclei of Arabidopsis cells. Overexpression of HDA19 in 35S:HDA19 plants decreased histone acetylation levels, whereas downregulation of HDA19 in HDA19-RNA interference (RNAi) plants increased histone acetylation levels. In comparison with wild-type plants, 35S:HDA19 transgenic plants had increased expression of ETHYLENE RESPONSE FACTOR1 and were more resistant to the pathogen A. brassicicola. The expression of jasmonic acid and ethylene regulated PATHOGENESIS-RELATED genes, Basic Chitinase and beta-1,3-Glucanase, was upregulated in 35S:HDA19 plants but downregulated in HDA19-RNAi plants. Our studies provide evidence that HDA19 may regulate gene expression involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis.

The stability of the Arabidopsis transcriptome in transgenic plants expressing the marker genes nptII and uidA.

The ATH1 Arabidopsis GeneChip from Affymetrix was used to search for transcriptome changes in Arabidopsis associated with the strong expression of transgenes regulated by constitutive promoters. The insertion and expression of the commonly used marker genes, uidA and nptII, did not induce changes to the expression patterns of the approximately 24 000 genes that were screened under optimal growth conditions and under physiological stress imposed by low temperatures. Approximately 8000 genes (35% of the Arabidopsis genome) underwent changes in gene expression in both wild-type and transgenic plants under abiotic stresses such as salt, dehydration, cold, and heat. This study provides detailed information on the extent of non-targeted or pleiotropic effects of transgenes on plants and shows that the transgenic and non-transgenic plants were equivalent in their global patterns of transcription. This information may help to extend our understanding and interpretation of the principle of substantial equivalence which is used as a first step in the biosafety evaluation of transgenic crops.

Analysis and use of the tobacco eIF4A-10 promoter elements for transgene expression.

The eIF4A gene family codes for proteins which unwind secondary structures of mRNA during translational initiation. The tobacco eIF4A-10 promoter is one of a few of constitutive promoters found in plants. Research was conducted to identify the proximal promoter elements and to evaluate the potential application of the promoter for regulating transgene expression in a range of crop plants. A large intron (892 bp) in the leader sequence was found to be dispensable for constitutive promoter activity and did not contribute to the overall performance of the promoter. Deletion analysis showed that the upstream region between -151 bp and -73bp relative to the transcriptional start site was essential for the high level of expression and the constitutive activity. The data indicated that the elements in this region may coordinate and compensate each other for the high levels of promoter expression. The downstream leader sequence also contained a strong quantitative enhancer element that was essential for the full activity of the eIF4A-10 promoter. The eIF-4A10 promoter was found to be active in a wide range of plant species and tissues indicating that it will be useful for the constitutive expression of transgenes in plants.

Evidence that the hexose-to-sucrose ratio does not control the switch to storage product accumulation in oilseeds: analysis of tobacco seed development and effects of overexpressing apoplastic invertase.

Wild-type tobacco (Nicotiana tabacum L.) seed development was characterized with respect to architecture and carbohydrate metabolism. Tobacco seeds accumulate oil and protein in the embryo, cellular endosperm and inner layer of the seed coat. They have high cell wall invertase (INV) and hexoses in early development which is typical of seeds. INV and the ratio of hexose to sucrose decline during development, switching from high hex to high suc, but not until most oil and all protein accumulation has occurred. The oil synthesis which coincides with the switch is mostly within the embryo. INV activity is greater than sucrose synthase activity throughout development, and both activities exceed the demand for carbohydrate for dry matter accumulation. To investigate the role of INV-mediated suc metabolism in oilseeds, genes for yeast INV and/or hexokinase (HK) were expressed under a seed-specific napin promoter, targeting activity to the apoplast and cytosol, respectively. Manipulating the INV pathway in an oilseed could either increase oil accumulation and sink strength, or disrupt carbohydrate metabolism, possibly through sugar-sensing, and decrease the storage function. Neither effect was found: transgenics with INV and/or HK increased 30-fold and 10-fold above wild-type levels had normal seed size and composition. This contrasted with dramatic effects on sugar contents in the INV lines.