ABSTRACT: Controversy regarding genetically modified (GM) plants and their potential impact on human health contrasts with the tacit acceptance; of other plants that were also modified, but not considered as GM products (e.g., varieties raised through conventional breeding such as mutagenesis). What is beyond the phenotype of these improved plants? Should mutagenized plants be treated differently from transgenics? We have evaluated the extent of transcriptome modification occurring during rice improvement through transgenesis versus mutation breeding. We used oligonucleotide microarrays to analyze gene expression in four different pools of four types of rice plants and respective controls: (i) a; gamma-irradiated stable mutant, (ii) the M1 generation of a 100-Gy gamma-irradiated plant, (iii) a stable transgenic plant obtained for production of an anticancer antibody, and (iv) the T1 generation of a transgenic plant produced aiming for abiotic stress improvement, and all of the unmodified original genotypes as controls. We found that the improvement of a plant variety through the acquisition of a new desired trait, using either mutagenesis or transgenesis, may cause stress and thus lead to an altered expression of untargeted genes. In all of the cases studied, the observed alteration was more extensive in mutagenized than in transgenic plants. We propose that the safety assessment of improved plant varieties should be carried out on a case-by-case basis and not simply restricted to foods obtained through genetic engineering. Experiment Overall Design: Plant Materials Experiment Overall Design: Two genetically stable Oryza sativa L. ssp. japonica lines: a gamma-irradiated ricemutant(cv. EstrelaA)and a well characterized transgenic rice line (cv. Bengal) were used as well as controls. The stable mutant was obtained in 1988 by gamma-irradiation, had already gone over 10 generations of self-pollination, and had a mature average height about 45 cm lower than the wild type. The stable transgenic line, which was already in the third generation of self-pollination after transformation, expresses a ScFV antibody (ScFvT84.66) against carcinoembryonic antigen, a well characterized tumor-associated marker antigen. Experiment Overall Design: We have also used two genetically unstable rice lines: the M1 generation of a 100-Gy gamma-irradiated line (98% survival after mutagenesis) and the T1 generation of an Agrobacterium-transformed transgenic line (both cv. Nipponbare) containing one copy of the BCBF1 gene driven by the AtRD29A promoter from Arabidopsis and one copy of the hpt II gene. We used seeds from the same self-pollinated panicle for control and irradiation/transgenesis. The unstable mutant line chosen for this experiment was the one showing a phenotype more similar to that of the nonirradiated control. Experiment Overall Design: In the case of the transgenic lines, stability was based on the stable Experiment Overall Design: inheritance of the introduced transgenes in the homozygous progeny. Regarding the mutagenized plants we have defined as genetically stable plants those that, after mutagenesis, had already gone through several cycles of self-pollination while maintaining the desirable traits. Experiment Overall Design: RNA Extraction and Microarrays Experiment Overall Design: Two pools of six whole seedlings were prepared for each condition under test, and RNA was isolated with the RNeasy Plant Mini Kit (Qiagen) following the manufacturerâ??s instructions. Total RNA was kept at -80°C and sent to the Affymetrix core facility (Instituto Gulbenkian de Ciência, Experiment Overall Design: Oeiras, Portugal), where quality-control analysis was carried out before cDNA synthesis from themRNA[with appropriate oligo(dT) primers], labeling (through synthesis of cRNA with incorporation of biotinylated ribonucleotide analogs), and hybridization to the GeneChip Rice Genome Array (Affymetrix). This array contains probes to query 51,279 transcripts representing two rice subspecies (48,564 japonica transcripts and 1,260 transcripts of indica subspecies). Experiment Overall Design: Data Analysis Experiment Overall Design: Microarrays data analysis was performed with Partek Genomics Suite software. Affymetrix CEL files were imported by using the Robust Multichip Average method, which involves four steps: background correction of the perfect match values, quintile normalization across all of the chips in the experiment, Log2 transformation, and median polish summarization. The logged data were used for hierarchical cluster analysis and statistical analysis. Experiment Overall Design: Hierarchical cluster analysis was performed by using Pearsonâ??s dissimilarity product moment correlation coefficient and Wardâ??s algorithm. Experiment Overall Design: For the identification of differentially expressed genes we used ANOVA Experiment Overall Design: and a false discovery rate with a 0.05 threshold.
