A comparative analysis of insertional effects in genetically engineered plants: considerations for pre-market assessments.

During genetic engineering, DNA is inserted into a plant's genome, and such insertions are often accompanied by the insertion of additional DNA, deletions and/or rearrangements. These genetic changes are collectively known as insertional effects, and they have the potential to give rise to unintended traits in plants. In addition, there are many other genetic changes that occur in plants both spontaneously and as a result of conventional breeding practices. Genetic changes similar to insertional effects occur in plants, namely as a result of the movement of transposable elements, the repair of double-strand breaks by non-homologous end-joining, and the intracellular transfer of organelle DNA. Based on this similarity, insertional effects should present a similar level of risk as these other genetic changes in plants, and it is within the context of these genetic changes that insertional effects must be considered. Increased familiarity with genetic engineering techniques and advances in molecular analysis techniques have provided us with a greater understanding of the nature and impact of genetic changes in plants, and this can be used to refine pre-market assessments of genetically engineered plants and food and feeds derived from genetically engineered plants.

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.

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.

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.