Evaluation of the effects of sugarcane processing on the presence of GM DNA and protein in sugar.

The Brazilian Sucro-energy Sector produces both energy, in the form of ethanol fuel, industrial steam and electricity, and sugar. Centro de Tecnologia Canavieira (CTC), the leading Brazilian sugarcane breeding company, has developed a pipeline of insect-protected sugarcane varieties to control sugarcane borer damage. The goal of this manuscript is to present the results of studies with three genetically modified (GM) sugarcane varieties and to evaluate the published literature regarding the possible presence of GM sugarcane DNA or protein in raw or refined sugar. Specifically, two varieties of approved GM sugarcane, CTC91087-6 and CTC175-A, and an experimental CTC variety, were grown in four individual plots to produce four batches each of processed raw sugar using standard smaller-scale laboratory processing methods resulting in a total of 12 independent batches of raw sugar. Herein, we report the development of event-specific probes and DNA detection methods, designed to detect the junction of sugarcane genomic DNA and the inserted DNA of the two approved GM varieties. An identical approach was used for the testing of sugar made from the experimental CTC variety. The methodology used TaqMan® real-time PCR and ELISA assays validated for the four GM proteins expressed by these three events (Cry1Ab, Cry1Ac, NPTII, and PAT (bar)). The developed assays had very low limits of detection (LODs) for the various event-specific DNA probes (7.2-25 ng/g sugar) and insecticidal and selectable marker proteins (2.9-10.9 ng/g sugar). No event-specific DNA and no GM proteins were detectable in the 12 independent batches of raw sugar produced from these three GM sugarcane events. The results of this study, using very sensitive methods and testing several sugar batches, extend the conclusions of previous studies, reviewed herein, that showed the extensive degradation and removal of DNA and protein during sugarcane processing. Overall, these results indicate that there are no distinguishable differences between the highly purified, chemically defined sugar produced from conventional or GM varieties.

Lack of Detection of Bt Sugarcane Cry1Ab and NptII DNA and Proteins in Sugarcane Processing Products Including Raw Sugar.

Brazil is the largest sugarcane producer and the main sugar exporter in the world. The industrial processes applied by Brazilian mills are very efficient in producing highly purified sugar and ethanol. Literature presents evidence of lack of DNA/protein in these products, regardless of the nature of sugarcane used as raw material. Recently CTNBio, the Brazilian biosafety authority, has approved the first biotechnology-derived sugarcane variety for cultivation, event CTC175-A, which expresses the Cry1Ab protein to control the sugarcane borer (Diatraea saccharalis). The event also expresses neomycin-phosphotransferase type II (NptII) protein used as selectable marker during the transformation process. Because of the high purity of sugar and ethanol produced from genetically modified sugarcane, these end-products should potentially be classified as "pure substances, chemically defined," by Brazilian Biosafety Law No. 11.105. If this classification is to be adopted, these substances are not considered as "GMO derivatives" and fall out of the scope of Law No. 11.105. In order to assess sugar composition and quality, we evaluate Cry1Ab and NptII expression in several sugarcane tissues and in several fractions from laboratory-scale processing of event CTC175-A for the presence of these heterologous proteins as well as for the presence of traces of recombinant DNA. The results of these studies show that CTC175-A presents high expression of Cry1Ab in leaves and barely detectable expression of heterologous proteins in stalks. We also evaluated the presence of ribulose-1,5-bisphosphate carboxylase/oxygenase protein and DNA in the fractions of the industrial processing of conventional Brazilian sugarcane cultivars. Results from both laboratory and industrial processing were concordant, demonstrating that DNA and protein are not detected in the clarified juice and downstream processed fractions, including ethanol and raw sugar, indicating that protein and DNA are removed and/or degraded during processing. In conclusion, the processing of conventional sugarcane and CTC175-A Bt event results in downstream products with no detectable concentrations of heterologous DNA or new protein. These results help in the classification of sugar and ethanol derived from CTC175-A event as pure, chemically defined substances in Brazil and may relieve regulatory burdens in countries that import Brazilian sugar.

Repulsive Guidance Molecules a, b and c Are Skeletal Muscle Proteins, and Repulsive Guidance Molecule a Promotes Cellular Hypertrophy and Is Necessary for Myotube Fusion.

Repulsive guidance molecules (RGMs) compose a family of glycosylphosphatidylinositol (GPI)-anchored axon guidance molecules and perform several functions during neural development. New evidence has suggested possible new roles for these axon guidance molecules during skeletal muscle development, which has not been investigated thus far. In the present study, we show that RGMa, RGMb and RGMc are all induced during skeletal muscle differentiation in vitro. Immunolocalization performed on adult skeletal muscle cells revealed that RGMa, RGMb and RGMc are sarcolemmal proteins. Additionally, RGMa was found to be a sarcoplasmic protein with a surprisingly striated pattern. RGMa colocalization with known sarcoplasmic proteins suggested that this axon guidance molecule is a skeletal muscle sarcoplasmic protein. Western blot analysis revealed two RGMa fragments of 60 and 33 kDa, respectively, in adult skeletal muscle samples. RGMa phenotypes in skeletal muscle cells (C2C12 and primary myoblasts) were also investigated. RGMa overexpression produced hypertrophic cells, whereas RGMa knockdown resulted in the opposite phenotype. RGMa knockdown also blocked myotube formation in both skeletal muscle cell types. Our results are the first to show an axon guidance molecule as a skeletal muscle sarcoplasmic protein and to include RGMa in a system that regulates skeletal muscle cell size and differentiation.

Response of sugarcane to increasing concentrations of copper and cadmium and expression of metallothionein genes.

Sugarcane (Saccharum spp.) offers the potential to be a phytoremediator species due to its outstanding biomass production, but its prospective metal accumulation and tolerance have not been fully characterized. Sugarcane plantlets were able to tolerate up to 100microM of copper in nutrient solution for 33 days, with no significant reduction in fresh weight, while accumulating 45mgCukg(-1) shoot dry weight. Higher levels of copper in solution (250 and 500microM) were lethal. Sugarcane displayed tolerance to 500microM Cd without symptoms of toxicity, accumulating 451mgCdkg(-1) shoot dry weight after 33 days, indicating its potential as Cd phytoremediator. DNA gel blot analyses detected 8 fragments using a metallothionein (MT) Type I probe, while 10 were revealed for the MT Type II and 8 for MT Type III. The number of genes for each type of MT in sugarcane might be similar to the ones identified in rice considering the interspecific origin of sugarcane cultivars. MT Type I gene appeared to present the highest level of constitutive expression, mainly in roots, followed by MT Type II, corroborating the expression pattern described based on large-scale expressed sequence tags sequencing. MT Type II and III genes were more expressed in shoots, where MT I was also importantly expressed. Increasing Cu concentration had little or no effect in modulating MT genes expression, while an apparent minor modulation of some of the MT genes could be detected in Cd treatments. However, the level of response was too small to explain the tolerance and/or accumulation of Cd in sugarcane tissues. Thus, cadmium tolerance and accumulation in sugarcane might derive from other mechanisms, although MT may be involved in oxidative responses to high levels of Cd. Sugarcane can be considered a potential candidate to be tested in Cd phytoremediation.