Subcellular location and transcriptional activation assay of MhERF113-like
To determine the subcellular localization of MhERF113-like, Nicotiana benthamiana leaves were transformed with a 35S: MhERF113-GFP construct. The results showed fluorescence signal for MhERF113-like was found in the nucleus and cell membrane, indicating that MhERF113-like was expressed in the nucleus and cell membrane of Nicotiana benthamiana leaves (Fig. 2A). To detect the transactivation activity of MhERF113-like, yeast two-hybrid (Y2H) assay were carried out with PGBKT7- MhERF113-like expressed in Y2H yeast cells. The results that pGBKT7-53 grew well and turned blue as a positive control in SD/-Ade/-His/-Leu/Trp + X-a-GAL medium, and the yeast cells transformed with pGBKT7-MhERF113-like also grew and turned blue on SD/-Ade/-His/-Leu/Trp + X-a-GAL medium, including different concentrations of AbA (Fig. 2B), suggesting that MhERF113-like has transcriptional activity.
Expression pattern analysis of MhERF113-like
To investigate the potential biological function of MhERF113-like, the expression levels of MhERF113-like under different stresses were determined by qRT-PCR with high expression in response to drought. Under drought stress, the expression level of MhERF113-like was the higher at 6 d, and its expression level was approximately 11-fold that of the control (Fig. 3A). The expression level of MhERF113-like showed an upward trend after 4 d of salt treatment (Fig. 3B). After ABA treatment, the expression level of MhERF113-like displayed a trend of first increasing and then decreasing (Fig. 3C). Moreover, MhERF113-like was most high expressed in the roots, followed in the stems (Fig. 3D). These results suggested that MhERF113-like may play critical role in response to drought stress.
MhERF113 -like positively regulates the drought tolerance of apple callus.
To identify the function of MhERF113-like in regulating drought tolerance in apple, the vector of MhERF113-like overexpression was constructed and transformed into apple ‘Orin’ callus, three MhERF113-like-OE lines (OE-1, OE-2, and OE-3) were screened by DNA and qRT-PCR identification (Fig. S1). Transgenic calluses were grown on MS medium supplied with 0 or 4% PEG6000 for drought tolerance assay (Fig. 4A). No differences in fresh weight were observed between the WT and OE calluses under the control condition. However, three OE calluses showed higher fresh weight than that of the WT under PEG treatment (Fig. 4B). To further support the results of the drought tolerance, we examined the electrolyte leakage and MDA content of the WT and OE calluses. The results showed electrolyte leakage and MDA contents in OE calluses were significantly lower than that of the WT under PEG treatment (Fig. 4C, D). Similarly, H2O2 content was significantly decreased in the OE callus under drought stress, compared to the WT callus (Fig. 4E). These results suggested that MhERF113-like positively regulated the drought tolerance of apple callus.
Ectopic expression of MhERF113-like enhances the drought tolerance in tomato
To further verify the function of MhERF113-like in drought tolerance, MhERF113-like was ectopically over-expressed in tomatoes. Three overexpression lines (OE#1, OE#9, and OE#12) with high expression levels of MhERF113-like were selected for drought tolerance assay (Fig. S2). At the beginning of the experiment (0 d), the WT and OE plants displayed similar phenotypes. However, after 6 days of drought, the WT plants displayed leaf wilting while the OE plants were essentially healthy. Subsequently, we found obvious differences after 12 days of drought, the OE plants displayed severe leaf wilting while the OE plants were less impacted (Fig. 5A). After recovery for 2 days, the recovery ability of the OE plants is stronger than that of the WT plants, which is consistent with the weaker chlorophyll fluorescence observed in WT leaves (Fig. 5D). Simultaneously, we examined the drought tolerance of the WT and OE plants in half-strength nutrient solution containing 4% PEG6000. The results that the wilting degree of the WT plants was more serious than that of the OE plants after exposure to PEG medium for 12 days, the result was consistent with that of soil drought treatment (Fig. S3). Consistent with this observation, detached leaves from the OE plants showed a lower water loss rate than leaves from the WT plants (Fig. 5C). These results indicated that the OE plants improved drought tolerance at least partly by minimizing water loss.
Overexpressing of MhERF113-like in tomato alleviates the oxidative damage
To characterize the effect of MhERF113-like overexpression on oxidative damage, the electrolyte leakage, MDA, H2O2, and O2− contents were measured. No significant differences in electrolyte leakage and MDA content between the WT and OE plants under the control condition. However, both WT and OE plants displayed a notable increase in electrolyte leakage and MDA content, and this increase was more in WT plants than that of OE plants under drought stress (Fig. 5E and F). In addition, there were no significant differences in H2O2 and O2− contents between WT and OE plants under control condition. However, H2O2 and O2− contents of the WT plants were significantly higher than those of the OE plants. The leaves were also histochemically stained with DAB and NBT to determine the accumulation of H2O2 and O2−, respectively. The staining intensity of DAB and NBT in the WT leaves was significantly greater than that of the OE leaves under drought stress (Fig. 5B). These results suggested that MhERF113-like resulted in less oxidative damage to the OE plants than to the WT plants under drought stress.
MhERF113 -like regulates the expression of the stress-related genes
To explore the potential molecular mechanisms of MhERF113-like in drought tolerance, the expression levels of stress-related genes, including SlDREB, SlRD29, SlAPX1, SlSOD, SlPOD, and SlCAT were analyzed between WT and OE tomatoes under control and drought conditions (Fig. 6). Under the control conditions, there were no significant changes in the expression levels of SlDREB, SlRD29, SlAPX1, SlSOD, SlPOD and SlCAT between WT and OE tomatoes. Under drought stress, the expression level of these genes, except for SlCAT and SlPOD, were upregulated in both the WT and OE tomatoes, and the upregulation of SlDREB and SlRD29 in tomato leaves was significantly higher in OE tomatoes than that of the WT plants. These results suggested that MhERF113-like may enhance plant drought tolerance by positively regulating the expression of stress response genes.