Figure 5. Transcriptional activation of SmNAC.

The SmNAC coding sequence without the stop codon was cloned into the reconstructed GAL4-DBD vector. To assay the binding activity of SmNAC to the ICS1 promoter, the ICS1 promoter was cloned into a pGreenII 0800-LUC double reporter vector, whereas SmNAC was inserted into the pGreenII 62-SK vector, generating the effector construct. The effector and reporter plasmids were co-transformed into S. melongena protoplasts as previously described and incubated as described above. LUC and REN luciferase activities were measured using a dual luciferase assay kit (Promega, USA). The analysis was performed using the Luminoskan Ascent Microplate Luminometer (Thermo, USA) with a 5 s delay and 15 s integration time. The binding activity of SmNAC to the ICS1 promoter was measured as a ratio of LUC to REN. At least six transient assays were measured for each assay. (a) The dual luciferase reporter construct contains a LUC reporter gene driven by the 35S (TATA box) promoter with five GAL4-binding elements, whereas each of the effectors contain a GAL4 DNA-binding domain (GAL4-BD); pBD was used as a negative control. SmNAC was linked to the GAL4-BD sequence and expression was driven by a 35S promoter. (b) Transactivation activity of SmNAC. Plasmid combinations of the dual REN/ LUC reporter, and effectors were co-transformed into eggplant protoplasts. After 12 h, the transactivation activity of SmNAC was measured as a ratio of LUC to REN. Each value represents the means of three biological replicates, and vertical bars represent the S.E. The asterisk indicates a significant difference at the 5% level.