Co-Expression of JcNAC1- and JcZFP8-Improved Agronomic Traits of Tobacco and Enhanced Drought Resistance through NbbHLH1 and NbbHLH2

Previous studies have identified numerous transcription factors involved in drought response, each of which play different roles in plants. The objective of the present study was to evaluate the effectiveness of two transcription factors on drought response in Jatropha curcas L., JcNAC1 and JcZFP8. The overexpression of these transcription factors in tobacco (Nicotiana benthamiana L.) improved drought resistance, but JcZFP8 delayed germination and JcNAC1 reduced biomass and yield. By constitutively co-expressing these two genes in tobacco, drought resistance was improved, and the negative effects of each of them were overcome. The transgenic plants with double-gene co-expression showed stronger drought tolerance with 1.76-fold greater accumulation of proline and lower H2O2 and malondialdehyde (MDA) content to 43 and 65% of wildtype (WT) levels, respectively. The expression levels of NbbHLH1 and NbbHLH2 genes upregulated linearly with the increased drought tolerance of double genes co-expression plants. In drought conditions, the leaf water contents of bhlh1, bhlh2, and bhlh1bhlh2 deletion mutants obtained by CRISPR-CAS9 knockout technique were maintained at 99%, 97%, and 97% of WT. The bhlh1bhlh2 was found with lower germination rate but with higher reactive oxygen levels (1.64-fold H2O2 and 1.41-fold MDA levels). Thus, the co-expression of two transcription factors with different functions overcame the adverse traits brought by a single gene and enhanced the shared drought-tolerant traits, which can provide guidance on theory and selection of gene combinations for the application of multi-gene co-expression in agriculture in the future.