Elevated CO2 alters transgene methylation not only in promoterregion but also in codingregion of Bt rice under different N-fertilizer levels

The earth has been undergoing climate change, especially in recent years, driven by increasing concentration of atmospheric carbon dioxide (CO 2 ) and rising earth-surface temperature, which could reduce N allocation to Bt toxin for transgenic Bt crops ( Bt crops), but the N fertilization is considered to be an effective method to enhance the C–N balance in Bt crops in the case of elevated CO 2 in future. DNA methylation not only in promoterregion but also in codingregion of transgene plays a critical role in transgene expression regulation and silencing of transgenic crops. Recent research has emphasized the risks of increased transgene silencing of Bacillus thuringiensis ( Bt ) rice under elevated CO 2 . In this study, the effects of elevated CO 2 (vs. ambient CO 2 ) on exogenous Bt toxins and transgene expression in promoterregion and codingregion of Bt rice during tillering stage (cv. HH1 expressing fused Cry1Ab/Cry1Ac ) were evaluated under three nitrogen (N) fertilizer rate (1/4, 1 and 2 N levels). The aboveground and belowground biomass, and foliar Bt protein content of Bt rice were all significantly increased with the augmentation of N-fertilizer. And elevated CO 2 significantly increased belowground biomass, total soluble protein content, transgene methylation levels in promoterregion (P1), and in total of promoterregion(P1) and codingregion (P2 + P3) (i.e., P1 + P2 + P3) at 1 N level, and it also increased transgene methylation levels in codingregion (P2), and in total of promoterregion and codingregion (P1 + P2 + P3) at 2 N level. In addition, elevated CO 2 decreased foliar Bt protein content at 1 N level. The transgene methylation levels in promoterregion and codingregion were negatively correlated with Bt -transgene expression level. The methylation level of cytosines located at CG sites was higher than those at CHG and CHH sites in P1, P2 and P3 fragments regardless of the CO 2 or N-fertilizer level. The correlation of transgene mehtylation in promoterregion with transgene expression is even stronger than that in codingregion. These data indicate that N fertilization supply will increase the Bt toxin content in transgenic Bt rice, especially under elevated CO 2 .