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.

Jatropha curcas is widely planted as a highly drought-resistant biodiesel feedstock. Curcin protein is one of the Jatropha ribosomal inactivation proteins with broad-spectrum antifungal activity that may enter the soil ecosystem as a result of large-scale Jatropha cultivation and affect fungi and various enzymatic activities in the soil. In this research, the influence of curcin protein and Jatropha planting on soil fungi was investigated, and the levels of curcin in various tissues and organs of Jatropha were measured with an enzyme-linked immunosorbent assay. It was found that the content of curcin in seed kernels reaches 2 mg/g, which is much higher than that in other tissues. After the seeds have fallen into the soil, the level of curcin in the soil rises rapidly, reaching 59.22 µg/g soil and 67.49 µg/g soil in different soil samples, respectively. It then falls by more than 99% within six days. High-throughput sequencing technology was used to study the soils treated with different concentrations of curcin, and the results of the soil fungal alpha diversity index analysis showed that the fungal communities did not change significantly, but the abundance of each fungal community changed significantly. The degree of influence of different concentrations of curcin treatment on the abundance of the soil dominant fungal community were investigated for concentrations of 0.5 μg/g, 50 μg/g and 5 μg/g, and showed that concentrations of 0.5 μg/g and 50 μg/g are more likely to change fungal community structure in soil, and with the increasing extension of the treatment time, they may be detrimental to the conservation of soil ecosystems. Internal transcribed spacer (ITS) sequencing of soil fungi from Jatropha planted and unplanted areas in four regions with different climate types showed that Jatropha planting significantly altered the soil fungal communities in each region. There was a negative impact on soil fungal communities in tropical maritime monsoon and subtropical dry and hot monsoon climates, while a positive impact was observed in subtropical monsoon and tropical highland monsoon climates due to Jatropha cultivation. In conclusion, Jatropha plantations and curcin protein have an impact on soil fungi and thereby affect the ecological system of the soil.