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 (CO2) 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 CO2 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 CO2. In this study, the effects of elevated CO2 (vs. ambient CO2) 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 CO2 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 CO2 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 CO2 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 CO2.


Results
Belowground and aboveground biomass of Bt rice. CO 2 , N-fertilizer levels and their interaction were significantly affected both the belowground and aboveground biomass of Bt rice (P < 0.05 or 0.001; Table 1). Both the belowground and aboveground biomass significantly increased with increased N-fertilizer augmentation, respectively (P < 0.05; Fig. 1). Compared with ambient CO 2 , elevated CO 2 significantly increased the aboveground biomass of Bt rice grown at 2 N-fertilizer level (+ 25.74%), and belowground biomass of Bt rice grown at 1 N and 2 N-fertilizer levels (+ 27.71% and + 21.19%; P < 0.05, Fig. 1).
Foliar contents of total soluble protein and Bt protein of Bt rice. N-fertilizer level significantly affected the foliar content of total soluble protein of Bt rice (P < 0.001; Table 1). Under ambient CO 2 , the foliar content of total soluble proteins of Bt rice grown at 1/4 N level were significantly lower (− 16.14%) than that at 2 N level (P < 0.05; Fig. 2A). Under elevated CO 2 , the foliar content of total soluble proteins of Bt rice grown at reduced N-fertilizer level (1/4 N) were significantly lower than that at 1 N and 2 N levels (− 17.27% and − Table 1. Two-way ANOVAs for the effects of CO 2 and N-fertilizer levels, and their interaction on the belowground and aboveground biomass, foliar contents of total soluble protein and Bt toxin, Bt-transgene expression and methylation in promoter and coding regions of Bt rice with fused Cry1Ab/Ac during tillering stage, grown under ambient and elevated CO 2 with different N-fertilizer levels (F and P values). Parameters CO 2 level (CO 2 ) N-fertilizer level (N) CO 2 × N    www.nature.com/scientificreports/ 15.70%; P < 0.05, Fig. 2A). Compared with ambient CO 2 , elevated CO 2 significantly increased the foliar content of total soluble proteins of Bt rice grown at 1 N level (+ 10.75%; P < 0.05, Fig. 2A). N-fertilizer level (P < 0.001) and its interaction with CO 2 level (P < 0.05) significantly influenced the foliar Bt protein content of Bt rice (Table 1). Under ambient CO 2 , the foliar Bt protein content of Bt rice significantly increased with the N fertilizer augmentation (P < 0.05; Fig. 2B). Under elevated CO 2 , the foliar Bt protein content of Bt rice grown at 2 N level was significantly higher than that at 1/4 and 1 N levels (+ 88.21% and + 61.47%; P < 0.05; Fig. 2B). Compared with ambient CO 2 , elevated CO 2 significantly decreased the foliar Bt protein content of Bt rice grown at 1 N level (− 16.04%; P < 0.05; Fig. 2B).

F-values P-values F-values P-values F-values P-values
Bt transgene expression in the leaves of Bt rice. N-fertilizer level (P < 0.05) and its interaction with CO 2 level (P < 0.001) significantly affected the Bt transgene expression in the leaves of Bt rice (Table 1). Under ambient CO 2 , the Bt-transgene expression level in the leaves of Bt rice grown at 1/4 N and 2 N level was significantly down-regulated when compared with that at 1 N level (− 38.16% and − 19.04%; P < 0.05; Fig. 3). Compared with ambient CO 2 , elevated CO 2 just significantly up-regulated the Bt-transgene expression level in the leaves of Bt rice grown at 1/4 N level (+ 48.03%; P < 0.05; Fig. 3).
Methylation status in the promoterregion and codingregion of Bt-transgene in the leaves of Bt rice. Promoterregion (P1) of Bt-transgene. CO 2 , N-fertilizer levels and their interaction significantly affected the methylation levels in the promoterregion (P1) of Bt-transgene in the leaves of Bt rice (P < 0.05; Table1). N-fertilizer level differently affected the methylation in the P1 fragment of Bt-transgene in the leaves of Bt rice. The methylation percentages in the P1 fragment of Bt-transgene in the leaves of Bt rice grown at 1/4 N level (+ 135.89%) and 2 N level (+ 157.23%) were markedly higher than that at 1 N level under ambient CO 2 , respectively (P < 0.05; Fig. 4), while it was contrary tendency under elevated CO 2 . Significant decreases in the methylation percentages were found in the P1 fragment of Bt-transgene in the leaves of Bt rice grown at 1/4 N level (− 62.52%) and 2 N level (− 33.75%) in contrast to that at 1 N level under elevated CO 2 (P < 0.05; Fig. 4). In addition, compared with ambient CO 2 , elevated CO 2 obviously decreased the methylation percentages in the P1 fragment of Bt-transgene in the leaves of Bt rice grown at reduced N-fertilizer level (1/4 N) (− 24.21%; P > 0.05), and markedly enhanced the methylation percentages in the P1 fragment of Bt-transgene in the leaves of Bt rice grown at recommended normal (1 N: + 376.96%; P < 0.05) and increased N-fertilizer level (2 N: + 22.84%; P > 0.05, Fig. 4). CO 2 , N-fertilizer levels and CO 2 × N-fertilizer interactions significantly affected the methylation levels of cytosines located at CG and CHH sites in the P1 fragment of Bt-transgene in the leaves of Bt rice (P < 0.05;  www.nature.com/scientificreports/ Table 2). The methylation levels of cytosines located at CHG site in the P1 fragment of Bt-transgene in the leaves of Bt rice was just significantly affected by CO 2 and CO 2 × N interactions (P < 0.05; Table 2). Under ambient CO 2 , the methylation level of cytosines located at CG and CHH sites in the P1 fragment of Bt-transgene in the leaves of Bt rice grown at 1/4 N level (+ 122.95% and + 140.32%; P < 0.05) and 2 N level (+ 112.82% and + 249.95%; P < 0.05) were markedly higher than that at 1 N level. In contrast, the methylation level of cytosines located at CG, CHG and CHH sites in the P1 fragment of Bt-transgene in the leaves of Bt rice grown at 1/4 N level (− 57.77%, − 58.41 and − 72.66%; P < 0.05) were significantly lower than that at 1 N level under elevated CO 2 (Fig. 5). Moreover, compared with ambient CO 2 , elevated CO 2 markedly enhanced the methylation percentages of cytosines located at CG, CHG and CHH sites in the P1 fragment of Bt-transgene in the leaves of Bt rice grown at 1 N level (+ 313.79%, + 397.40% and + 511.32%; P < 0.05), and CG sites in the P1 fragment of Bt-transgene in the leaves of Bt rice grown at increased N-fertilizer level (2 N: + 35.67%; P < 0.05) (Fig. 5). The methylation level of cytosines located at CG sites was higher than those at CHG and CHH in the P1 fragment of Bt-transgene in the leaves of Bt rice regardless of the CO 2 or N-fertilizer level (Fig. 5).
Codingregion (P2, P3, P2 + P3) of Bt-transgene. The interaction between CO 2 and N-fertilizer levels significantly affected the methylation levels in the codingregion (P2 + P3) of Bt-transgene in the leaves of Bt rice (P < 0.05; Table 1). Compared with ambient CO 2 , elevated CO 2 significantly enhanced the methylation percentages in the P2 + P3 fragments of Bt-transgene in the leaves of Bt rice grown at increased N-fertilizer level (2 N: + 47.24%; P < 0.05) (Fig. 6). CO 2 × N-fertilizer interaction significantly affected the methylation levels of cytosines located at CG site in the P2 + P3 fragments of Bt-transgenein the leaves of Bt rice (P < 0.05; Table 2). Compared with ambient CO 2 , elevated CO 2 significantly enhanced the methylation percentages of cytosines located at CG sites in the P2 + P3 fragments of Bt-transgene in the leaves of Bt ricegrown at 2 N level (+ 67.52%; P < 0.05; Fig. 5).
The methylation level of cytosines located at CG sites was higher than those at CHG and CHH sites in the P2 + P3 fragments of Bt-transgene in the leaves of Bt rice regardless of the CO 2 or N-fertilizer level (Fig. 5). CO 2 , Nitrogen-fertilizer levels and their interaction did not significantly affect methylation levels in the codingregion (P2) and codingregion (P3) of Bt-transgene in the leaves of Bt rice (P > 0.05; Table 1). In the codingregion (P2), the methylation percentage at 2 N level under elevated CO 2 (16.28%) was significantly higher than that under ambient CO 2 (9.72%) (P < 0.05, Fig. 6). There were no CHG and CHH sites as potential targets in the P2 fragment of Bt-transgene (Fig. 5). In the codingregion (P3), the methylated level was very low, not exceeding 5.74% (Fig. 6). The methylation level in the P3 fragment was lower than that in the P2 fragment of Bt-transgene in the leaves of Bt rice (Fig. 6). N-fertilizer level significantly influenced the methylation level of cytosines located at CG site in the P3 fragment of Bt-transgene in the leaves of Bt rice (P < 0.05; Table 2). Under ambient CO 2 , methylation level of cytosines located at CG sites in the P3 fragment of Bt-transgene in the leaves of Bt rice grown under increased N-fertilizer level (2 N) was significantly lower that at 1 N level (− 70.47%, P < 0.05; Fig. 5).
Bt-transgene (P1 + P2 + P3). CO 2 and its interaction with N-fertilizer significantly affected the methylation levels in the Bt-transgene (P1 + P2 + P3) in the leaves of Bt rice (P < 0.001; Table 1). The methylation percentages in the P1 + P2 + P3 fragments of Bt-transgene in the leaves of Bt rice grown at 1/4 N level were significantly lower than that at 1 N and 2 N level under elevated CO 2 respectively (− 37.10% and − 15.80%; P < 0.05, Fig. 7). In addition, compared with ambient CO 2 , elevated CO 2 markedly enhanced the methylation percentages in the P1 + P2 + P3 fragments of Bt-transgene in the leaves of Bt rice grown at recommended normal (1 N: + 87.17%; P < 0.05) and increased N-fertilizer level (2 N: + 36.17%; P > 0.05) (Fig. 7). CO 2 , N-fertilizer levels and their interactions significantly affected the methylation levels of cytosines located at CHH sites in the P1 + P2 + P3 fragments of Bt-transgene in the leaves of Bt rice (P < 0.05; Table 2). The methylation levels of cytosines located at CG in the P1 + P2 + P3 fragments of Bt-transgene in the leaves of Bt rice was significantly affected by CO 2 and its interaction with N-fertilizer (P < 0.05; Table 2), while the methylation levels of cytosines located at CHG sites in the P1 + P2 + P3 fragments of Bt-transgene in the leaves of Bt rice was just significantly affected by CO 2 level. The methylation level of cytosines located at CG sites was higher than those at CHG and CHH sites in the P1 + P2 + P3 fragments of Bt-transgene in the leaves of Bt rice regardless of the CO 2 or N-fertilizer level (Fig. 5). Under ambient CO 2 , the methylation level of cytosines located at CHH sites in the P1 + P2 + P3 fragments of Bt-transgene in the leaves of Bt rice grown at 2 N level were markedly higher than that at 1 N level (+ 128.29%; P < 0.05, Fig. 5). Under elevated CO 2 , the methylation level of cytosines located at CG and CHH sites in the P1 + P2 + P3 fragments of Bt-transgene in the leaves of Bt rice grown at 1/4 N level were significantly lower than that at 1 N level respectively (− 33.79% and − 61.01%; P < 0.05, Fig. 5). In addition, compared with ambient CO 2 , elevated CO 2 markedly enhanced the methylation percentages of cytosines located at CG, CHG and CHH sites in the P1 + P2 + P3 fragments of Bt-transgene in the leaves of Bt rice grown at 1 N level (+ 62.03%, + 284.85% and + 229.98%; P < 0.05) and at CG sites in the P1 + P2 + P3 fragments of Bttransgene in the leaves of Bt rice grown at increased N-fertilizer level (2 N: + 53.77%; P < 0.05) (Fig. 5).
The correlation between the transgene methylation in promoterregion and codingregion, and the Bt-transgene expression level. The Pearson's analysis showed that the methylation level in the promoterregion (P1) of Bt-transgene was negatively correlated with the Cry1Ab/1Ac expression level in the leaves of Bt rice (Fig. 8). The methylation level in the codingregion (P2 + P3) was slightly negatively correlated with the Cry1Ab/1Ac expression level in the leaves of Bt rice (Fig. 8). The methylation level in the Bt-transgene (P1 + P2 + P3) was negatively correlated with the Cry1Ab/1Ac expression level in leaves of Bt rice during tillering stage (Fig. 8

Discussion
Previous studies showed that elevated CO 2 can stimulate plant growth and increase photosynthetic rate, photosynthate production, biomass and C: N ratios 36 . Hao et al. reported that the biomass of leaf, stem, pod, and total aboveground biomass of soybean increased with elevated CO 2 37 . Our results indicated that elevated CO 2 and increased N-fertilizer both increased the biomass of Bt rice. Also, it appeared that elevated CO 2 showed a positive  www.nature.com/scientificreports/ effect on the aboveground biomass of Bt rice grown under higher N-fertilizer (i.e., 2 N level) and belowground biomass of Bt rice grown under 1 N and 2 N-fertilizer. The biomass of Bt rice was significantly increased with increased augmentation of N fertilizer. It is expected that the increased nitrogen uptake by the plant would enhance the rate of photosynthesis, resulting in increased biomass accumulation via increased CO 2 diffusion conductance and Rubisco content in Bt rice leaves [38][39][40] . Hence, elevated CO 2 and augmentation of N supply simultaneously increased the rice biomass, likely manifesting synergistically additive effects on biomass accumulation. In recent years, the potential impacts of future CO 2 levels on Bt crops have attracted increasing attention. Our results show that foliar Bt protein content of Bt rice grown at elevated CO 2 were significantly lower than that under ambient CO 2 at 1 N level. It may be related to the decreased N allocation to Bt protein caused by elevated (P1, CpG island 1 (promoterregion); P2, CpG island 2 (codingregion); P3, CpG island 3 (codingregion); P2 + P3, CpG island 2 + CpG island 3 (codingregion); P1 + P2 + P3, CpG island 1 + CpG island 2 + CpG island 3 (Bttransgene)). Table 2. Two-way ANOVAs for the effects of CO 2 and N-fertilizer levels, and their interaction on the cytosine methylation percentage in the promoterregion (P1) and codingregion (P2, P3, P2 + P3) of Bt-transgene (P1 + P2 + P3) in the leaves of Bt rice with fused Cry1Ab/Ac during tillering stage, grown under ambient and elevated CO 2 with different N-fertilizer levels (F and P values).  22 . In this study, the foliar Bt toxin content of Bt rice at 2 N level was significantly higher than those at 1 N and 1/4 N level, indicating that the doubling of nitrogen augmentation (i.e., 2 N) resulted in the enhanced foliar Bt protein content level in the leaves of Bt rice. Bruns and Abel reported that the Bt protein production of two transgenic Bt-transgenic maize lines increased with the augmentation of N fertilizer application 41 . Yang et al. found that the contents of Cry2A and Cry1C in Bt rice both increased in the tillering and milking stages with the higher N concentrations applied on rice 42 . Wang et al. documented that the Cry1Ab/1Ac content of Bt-SY63 at higher N fertilizer was significantly higher than that without N fertilizer treatment 43 . Moreover, the foliar content of total soluble protein at 1/4 N level was significantly lower than that at 1 N and 2 N level, respectively. The Bt protein content in plant tissues has been shown to significantly correlate with soluble protein and overall nitrogen content 41,44 . Hence, it is plausible to increase the Bt protein content in Bt crops by taking appropriate nitrogen management measures.

F-values P-values F-values P-values F-values P-values
Epigenetic changes in DNA methylation can affect transgene expression for transgenic crops. DNA methylation occurs in codingregion has a more complex association with gene expression, whereas DNA methylation in promoterregion plays a vital role in transgene silencing 35 . For example, the resistance marker expression of transformed tobacco cultivars was rapidly lost and transgene expression were down-regulated, and hypermethylation within the 35S and NOS-promoters of these cultivars were found 45 . Additionally, environmental factors, such as drought and extreme temperature can potentially influence the methylation status [46][47][48] . In rice, 70% of the drought-induced methylation changing sites were reversed to their original status after water recovery 49 . In this study, our results showed that elevated CO 2 significantly enhanced the methylation percentages in the promoterregion (P1), and the P1 + P2 + P3 fragments of Bt-transgene in the leaves of Bt rice during tillering stage grown at 1 N level. In the codingregion, the methylation level in the P2 fragment of Bt-transgene, the fagment near the top strand of Bt-transgene, was higher than that in the P3 fragment, the fragment amplified from the bottom strand of Bt-transgene. Though the methylation level was low in P3 fragment of Bt-transgene, it was negatively correlated with the Cry1Ab/1Ac expressi in the leaves of Bt rice during tillering stage. In general, the methylation status in codingregion in Bt-transgene was slightly negatively correlated with the Cry1Ab/1Ac expression level in the leaves of Bt rice during tillering tage. Jiang et al. found that the PTGS methylation in the codingregion of Bt-transgene in the leaves of Bt rice during seeding stage remained at a relatively low level, lower than 5% 19 . The methylation level in the codingregion of Bt-transgene shows a weak regulation to the transgene expression. Thus, the methylation level in codingregionof Bt-transgene in the leaves of Bt rice has a weak regulation to the transgene expression both in tillering and seeding stage. The methylation levels in the promoterregion likely affected transgene expression more than that in the codingregion of Bt-transgene in the leaves of Bt rice. In addition, the Pearson's analysis also showed that the methylation level in the P1 + P2 + P3 fragments of Bt-transgene was negatively correlated with the Cry1Ab/1Ac expression in the leaves of Bt rice. Thus, the methylation level in the P1 + P2 + P3 fragments of Bttransgene was showed moderate regulation to the transgene expression in the leaves of Bt rice during tillering stage.
Stable transgene expression and heritability are key factors for the development and application of transgenic crops. Environmental factors, such as soil salinity, water accessibility and temperature all play crucial roles in Bt transgene expression 50,51 . Trtikova et al. found that the Cry1Ab expression in MON 810 maize under hot/ dry stress was significantly lower than that under optimal conditions 52 . Other studies with Bt crops have also indicated that environment might influence the levels of transgene expression differently 53 . Our results indicated that the Bt transgene expression was significantly up-regulated by elevated CO 2 under 1/4 N level, and Bt transgene expression level in the leaves of Bt rice grown at 1/4N and 2N level was significantly down-regulated when compared with that at 1N level under ambient CO 2 . Considering the methylation level in promoterregion and codingregion of Bt-transgene was negatively correlated with the Cry1Ab/1Ac expression level in the leaves of Bt rice during tillerage stage, so we speculate that the different transgene expression level among different CO 2 and N treatments was caused by methylation in promoterregion and codingregion of Bt-transgene and post-transcriptional regulation in the leaves of Bt rice during tillering stage.
In conclusion, the methylation level in the promoterregion and codingregion of Bt-transgene were negatively correlated with the Bt transgene expression level in the leaves of Bt rice during tillering stage. The methylation levels in the promoterregion likely affected transgene expression more than that in the codingregion of Bttransgene in the leaves of Bt rice during tillering stage. Elevated CO 2 showed positively effect on the transgene methylation level and negatively effect on the foliar Bt toxin content of Bt rice grown under 1 N level. The increased N-fertilizer level showed positively effect on the foliar Bt toxin content of Bt rice during tillering stage. Under elevated CO 2 situation in the future, moderate application of N-fertilizer can increase the foliar Bt toxin content in Bt rice. Futhermore, additional studies should be performed to evaluate the efficacy of the transgenic proteins against the target organisms under elevated CO 2 , and thus the biological meaning behind it.

Materials and methods
Plant materials. The Bt rice cultivar HH1 (Huahui 1) was used in the study. The rice seeds were provided by Prof. Yongjun Lin from Huazhong Agricultural University (Wuhan, China). HH1 was developed by using MH63 as the recipient to harbor the fusion gene Cry1Ab/Ac from transgenic event TT51-1 (GenBank Accession Number: EU880444.1). Expression of the Cry1Ab/Ac gene is driven by the rice actin 1 promoter and the nopaline synthase (NOS) gene terminator (seen in Fig. 9).
Plant growth conditions. This experiment was performed in electronically controlled growth incubator (GDN-400D-4/CO 2 ; Ningbo Southeast Instrument CO., LTD, Ningbo, China) connected with a gas-tank system for maintaining the desired atmospheric CO 2 concentration. The conditions in the chambers were maintained at 28 °C (day) and 25 °C (night) under a 16: 8 h light/dark photoperiod. The light intensity was 20,000 lx. Two CO 2 concentrations levels were applied continuously, i.e., elevated CO 2 (800 ppm, predicted CO 2 concentration Scientific Reports | (2020) 10:18138 | https://doi.org/10.1038/s41598-020-75121-6 www.nature.com/scientificreports/ in 2100), and ambient CO 2 (about 400 ppm). With each CO 2 level, the N-fertilizer was set at three levels, 1/4, 1 and 2 N; the 1 N was 1.25 mM NH 4 NO 3 . Therefore, the experiment was consisted of 2 CO 2 concentrations × 3 N-fertilizer levels (total 6 treatment combinations) deployed in six electronically controlled growth incubators as three replications for CO 2 main factors. The rice seeds of Bt rice (cv. HH1) were soaked in water for one day, and germinated on a board covered with wet cotton gauze for one day. Then, these seeds were sown into plastic foam covering (0.6 cm thick) on plastic cups (9 cm diameter, 7 cm height) and placed in the electronically controlled growth incubators of ambient and elevated CO 2 . In the cup, there were two holes in the plastic foam and one rice seeds into each hole (total two seeds per cup). Thirty cups were placed in each electronically controlled growth chambers with 10 cups per N-fertilizer level. The cups were filled with modified culture solutions 54

Measurement of plant biomass.
After sixty-five days for Bt rice grown under ambient and elevated CO 2 with different N-fertilizer levels (i.e., tillering stage), ten Bt rice plants for each N-fertilizer level were randomly selected from each growth incubator (i.e., 30 rice plants for each fertility-fertilizer level per CO 2 level). The biomass of belowground (root) and aboveground (stem and leaves) plant tissues were individually weighted with an electronic balance (Mettler Toledo AL 104; readability = 0.1 mg, repeatability < ± 0.1 mg).

Measurement of foliar contents of total soluble protein and Bt protein.
After the measurement of plant biomass, the foliar contents of total soluble protein and Bt protein in the sampled rice plants were measured using the diagnostic kit, A045-2 (Nanjing Jiancheng Bioengineering Institute) and ELISA kits from EnviroLogix (Portland, ME; catalog number AP003), respectively. Three leaves from each sampled plant were taken as a sample unit and weighed. Five samples were measured for each treatment. The samples were individually placed into 2 ml microreaction tubes and homogenized in a Tissue Lyser II (Qiagen) by shaking for 3 min at 30 Hz with two steel balls in each tube. For the determination of foliar total soluble protein content, 0.9% saline was used as an extraction buffer in a proportion of 1:9 (m/v). Then, the measurement was performed by following the kit instructions. Optical density (OD) values were measured using a UV-Vis spectrophotometer (UV-1800PC, Mapada, Shanghai, China) at 595 nm wavelength. For the determination of foliar Bt protein content, samples were mixed with extraction buffer PBST (provided with the kit) in a proportion of 1: 10 to 1: 100 (m/v) and then measured the foliar Bt protein content in the leaves of Bt rice during tillering stage according to the kit instructions. The OD values were measured using a UV-Vis spectrophotometer at 450 nm wavelength.

Bioassay of the transcript expression levels of Bt-transgene. RNA extraction and reverse tran-
scription. One leaf per rice plant was excised from 3 plants (total 3 leaves per replication) of each treatment combination of CO 2 and N-fertilizer levels for quantification of transcript expression levels of Bt-transgene in the leaves of Bt rice during tillering stage. Three samples were measured for each treatment. Total RNA was extracted from leaf tissues using TRIzol reagent following the supplier's protocol (Invitrogen). RNA concentration and integrity were evaluated using the NanoDrop spectrophotometer (Thermo Scientific). First strand cDNA templates were synthesized using Prime Script RT reagent kit (TaKaRa, Japan).
Real-time PCR analysis. Quantitative real-time PCR (qRT-PCR) experiment was carried out using SYBR Premix Ex Taq (TaKaRa, Japan) following the kit instructions. Expression of the target gene (i.e., Bt-transgene) was normalized relative to the expression of the housekeeping genes actin1 and ubiquitin. Quantification of the transcript level of Bt-transgene in the leaves of Bt rice during tillering stage was based on the method of Livak and Schmittgen 55 . Primers used for qRT-PCR are listed in Table 3.
Methylation analysis of Bt-transgene. Genomic DNA were extracted and purified from 30 mg treated leaves of Bt rice from each treatment combination of CO 2 and N-fertilizer levels during tillering stage using DNAsecure Plant Kit (TIANGEN, Beijing, China) following the product instructions. DNA concentration was quantified in the NanoDrop spectrophotometer. Then, 100 ng of isolated DNA was submitted to bisulfite treat- www.nature.com/scientificreports/ ment to convert non-methylated cytosines into uracil. The conversion was performed using the DNA Bisulfite Conversion Kit (TIANGEN, Beijing, China). Three types of cytosines -CG, CHG and CHH were analyzed in two regions of transgene: a fragment of the Actin 1 promoter (P1, CpG island 1) and two fragments of Cry1Ab/1Ac coding region (P2, CpG island 2 and P3,CpG island 3) ( Table 4). The bisulfite sequencing primers were designed using Methyl Primer Express Software (Applied Biosystems) ( Table 5). The target sequences of Bt-transgene were amplified from the Bisulfite-treated genomic DNA by PCR with Methylation-specific Kit (TIANGEN, Beijing, China). The PCR conditions consisted of denaturation at 95 °C for 5 min, followed by 35 cycles at 94 °C for 20 s, 60 °C for 30 s, 72 °C for 20 s, and annealing at 72 °C for 5 min. The PCR products were purified using AxyPrep DNA Gel Extraction Kit (Axygen, Union City, USA), cloned into pEASY-T3 Cloning Vector and transformed into Trans 1-T1 Phage Resistant Chemically Competent Cell (TransGen, Beijing, China). Positive clones were screened with PCR using M13R and M13F primers. Sequencing were done for at least ten independent positive clones from each PCR product was carried out.
Data analysis. All statistical analyses were conducted using SPSS (version 22.0; SPSS Inc., Chicago IL, USA; https ://www.ibm.com/produ cts/spss-stati stics ). DNA methylation levels (%) in CG, CHG and CHH cytosine types were assessed using the kismeth web tool. Two-way analysis of variances (ANOVAs) were performed to examine the effects of CO 2 (Ambient vs. Elevated) and N-fertilizer (1/4, 1 and 2 N), and their interactions on plant biomass, foliar contents of total soluble protein and Bt protein, the gene expression levels of Cry1Ab/Ac, and the methylation level in the promoterregion (P1) and codingregion (P2, P3, P2 + P3) of Bt-transgene (P1 + P2 + P3) in the leaves of Bt rice during tillering stage. If there were significant effects of CO 2 level, N-fertilizer  Table 4. DNA sequences of CpG islands in the protmoterregion (P1) and coddingregion (P2 and P3) of Bttransgene in the leaves of Bt rice during tillering stage, grown under ambient and elevated CO 2 with different N-fefrtilizer levels. www.nature.com/scientificreports/ level or their interaction, the least significant difference (LSD) test was used to separate the treatment means at P < 0.05. The Pearson's test was performed by R software (version R i386 3.4.2; https ://www.r-proje ct.org/) to analyze correlations among methylation level in promoter region and coding region of Bt-transgene with the transgene expression level in the leaves of Bt rice during tillering stage, grown under ambient and elevated CO 2 with different N-fertilizer levels.