*2.8. MdSAP15 Overexpression Enhances Osmotic Stress Tolerance by Arabidopsis Seedlings*

Since the transcription of *MdSAP15* mRNA was significantly accumulated under drought stress, we chose this gene for investigating its biological functions in *Arabidopsis*. After kanamycin-resistance screening and PCR detection using *Arabidopsis* genomic DNAs as templates, more than five transformants were identified and confirmed, with elevated levels of *MdSAP15* transcripts (Figure 7A). From these, we selected three transgenic lines (L1, 4, and 5) with high *MdSAP15* expression to evaluate its potential functioning in response to osmotic and drought stresses. For the osmotic stress assay, five-day-old seedlings grown on Murashige and Skoog (MS) agar plates were vertically plated on an MS agar medium supplemented with 0, 200, or 300 mM of mannitol. While the roots of "Col" and *MdSAP15* OE seedlings displayed similar growth characteristics on MS agar medium plates (Figure 7B), their growth was affected when treated with different concentrations of mannitol. For example, the primary roots and fresh weights of the transgenics were longer and heavier than those of the wild-type (WT) "Col" upon exposure to 200 or 300 mM of mannitol (Figure 7C,D).

**Figure 7.** Overexpression of *MdSAP15* in *Arabidopsis* increased osmotic tolerance in response to mannitol treatment. (**A**) PCR identification and relative expression analysis of *MdSAP15* in Col-0 wild-type (Col) and transgenic *Arabidopsis* lines. M, DNA marker; −, negative control (H2O); +, positive control (plasmid DNA of *35S::MdSAP15* pRI101AN vector). Specific primers for *MdSAP15* were used to detect relative expression levels of Col and transgenic *Arabidopsis* lines. (**B**) Representative images of "Col" and transgenic seedlings at 5 days after seeds had been cultivated for 11 days on MS medium alone (0 mM mannitol) or MS medium supplemented with 200 or 300 mM of mannitol. Bars = 1 cm. (**C**) Primary root lengths and (**D**) fresh weights of "Col" and transgenic seedlings measured on Day 5 after plants had been exposed for 11 days to osmotic stress. (**E**) Relative electrolyte leakage, and levels of chlorophyll (**F**), proline (**G**), and malondialdehyde (MDA) (**H**) in "Col" and transgenic seedlings, measured on Day 5 following treatment on MS medium with 0 mM, 200 mM, or 300 mM of mannitol for 11 d. Error bars represent SD based on three independent replicates. For (**C**–**H**), bars not labeled with same letters in each panel indicate values are significantly different at *p* < 0.05, based on one-way ANOVA and Duncan's tests.
