*4.7. Generation of Transgenic* Arabidopsis *and Its Phenotype under Stress Treatment*

Plant expression vector pBI121-*ZmWRKY106* was constructed as previously described [54], and was transformed to wild-type (WT) *Arabidopsis* using the *Agrobacterium*-mediated floral dip method. Columbia-0 (WT) was used for exogenous expression of *ZmWRKY106*. The transformed seeds were selected on MS medium containing 50 mM Kanamycin at 22 ◦C with a photoperiod of 16 h light/8 h dark (60% humidity) to obtain the positive plants. Three T<sup>3</sup> generation overexpression lines (*OE-ZmWRKY106-1*, *OE-ZmWRKY106-2*, *OE-ZmWRKY106-3*) with higher expression levels of *ZmWRKY106* were selected by qRT-PCR for further analysis. *Arabidopsis* seeds were grown as described previously [50]. Four-week-old seedlings of transgenic and WT *Arabidopsis* were collected at 0, 4, 12, 24 and 36 h after drought treatment (dried on filter paper) to examine the expression of stress-related genes by qRT-PCR. The specific primers of stress-related genes are listed in Supplementary Table S1. Three biological replicates were performed for qRT-PCR.

For the germination assay, WT and transgenic *Arabidopsis* seeds were placed on MS medium and MS medium supplemented with 4% (*w*/*v*) or 8% PEG6000. When the radicle had emerged from the seed coat, we considered the seed germinated. Seed germination was followed for five days, and the germination rate was analyzed. For the root growth assay, five-day-old seedlings were transferred to MS medium with or without 6% and 8% PEG6000 for seven days, and then root lengths were measured. Each treatment contained three independent replicates.

For high-temperature stress assay, five-day-old seedlings were placed at 45 ◦C for 5 h and then resumed growth at 22 ◦C as described previously [18]. After growing under normal conditions for seven days, we took photos and analyzed the survival rate. Each treatment contained three independent replicates. Values are means ± SD and statistically significant differences were based on the Student's test.
