# Overexpression of _PtDXS_ Enhances Stress Resistance in Poplars
## 2 Results
### Expression Level of PtDXS under Abiotic Stress  
First, 200 mM NaCl, 200 \(\upmu\)M abscisic acid (ABA), 10% PEG6000, and 2 mM hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) were chosen for introducing to 2-month-old seedlings of _P. trichocarpa_ grown on sterile half-strength MS medium (pH 5.8). QRT-PCR showed that the expression of _PtDXS_ was upregulated by 200 mM NaCl, 200 \(\upmu\)M ABA, 2 mM H\({}_{2}\)O\({}_{2}\), and 10% PEG6000. Treatment with 200 \(\upmu\)M ABA significantly upregulated the expression level of _PtDXS_ from 3 to 48 h, with a peak at 12 h (Figure 4B). Treatment with 2 mM H\({}_{2}\)O\({}_{2}\) caused to upregulate the _PtDXS_ expression significantly after 3 h, which persisted for 48 h with a peak at 8 h (Figure 4C). Treatment with 200 mM NaCl downregulated the expression of _PtDXS_ at 1 and 3 h, followed by an increase from 6-48 h with a peak at 48 h (Figure 4D). Treatment with 10% PEG6000 downregulated the expression of _PtDXS_ atl to 2 days, and the expression of _PtDXS_ increased subsequently, and reached the maximum on the 7 th day. (Figure 4E). These data suggested that PtDXS protein might play a role in the response to abiotic stresses. In addition, _PtDXS_ gene  
Figure 4: Comparison of _PtDXS_ expression in various tissues and in response to stresses. (**A**) Comparison of the _PtDXS_ expression in the indicated tissues. Comparison of the _PtDXS_ expression level in young leaves followed by different treatments and times (**B**) 200 \(\upmu\)M ABA, (**C**) 2 mM H\({}_{2}\)O\({}_{2}\), (**D**) 200 mM NaCl, and (**E**) 10% PEG6000. All experiments have been normalized by _gactin_ as an internal reference; bars represent means \(\pm\) standard deviation (SD; \(n=3\)); three independent biological experiments were performed with three repeats; asterisks represent significant differences in comparing with 0 h treatment as the control (Student’s \(t\)-test; \(\lx@note{footnote}{$*$}\), \(\lx@note{footnote}{$*$}\), and \(\lx@note{footnote}{$*$}\), \(p<0.05\), \(p<0.01\), and \(p<0.001\), respectively). significantly revealed higher expression in responding to H2O2 stress along certain time points 6, 24 and 48 h in comparing with the other stresses and times (Figure S4). Overexpression of PtDXS in Nanlin895 Poplar and Response of Transgenic Poplars to 100 mM NaCl and 3% PEG6000 Stresses  
To analyze the _PtDXS_ functional roles in transgenic poplars, we used the Agrobacteriumium-mediated transformation to overexpress _PtDXS_ gene into WT poplars. Twelve independent transgenic poplars were achieved through screening of regenerated kanamycin-resistant poplars (Figure S5). PCR was performed to determine whether T-DNA was inserted into the genome of Nanlin895 poplars, and qRT-PCR and western blot were used to confirm expression of the target gene (Figure S6). When we used the 35S primer as the upstream primer and _PtDXS_-R primer as the downstream prime, the genomic PCR analyses illustrated that the twelve transgenic lines had the expected bands, but the wild-type poplars had no bands according to the analysis of 1% agarose gel electrophoresis (Figure S6A). In PCR reactions, the _PtDXS_-F and _PtDXS_-R primers isolated one longer fragment > 2166 bp in WT poplars, because of introns, whereas expected targeted bands were appeared in the transgenic lines (Figure S6B). In addition, the result of PCR for _npt_II (kanamycin resistance) gene showed that the twelve transgenic lines exhibited expected bands, but the wild-type poplars exhibited no bands (Figure S6C). The result of southern blot revealed that 2-4 copies of _PtDXS_ gene were overexpressed in transgenic lines (Figure S6D). The twelve transgenic lines also analyzed by qRT-PCR to show the _PtDXS_ transcript levels. qRT-PCR result showed that _PtDXS_ gene were expressed in Nanlin895 poplars (Figure S6E). Total protein from WT and transgenic plants was extracted and analyzed by 12% SDS-PAGE (Figure S6F). Furthermore, the results of western blot revealed the presence of PtDXS expressed peptides using His-DXS.