Heat and drought stress can cause severe negative impacts on canola (mainly B. napus). The targeted identification of specific genetic determinants of stress adaptation and generation of related canola lines are effective approaches in breeding cultivars with strong stress resistance. Since canola’s value chain is centered largely on seed oil, it is attractive to simultaneously improve abiotic stress tolerance and increase/maintain seed yield and quality (oil content, fatty acid profile, etc.). The overall objective was to evaluate canola lines with distinct modifications in Brassica napus phospholipid: diacylglycerol acyltransferase (BnPDAT1), an enzyme catalyzing the last step in triacylglycerol biosynthesis, under heat and drought stress at the flowering stage, and to use transcriptomic analysis to identify additional candidate genes related to heat and drought stress. The specific objectives were to (1) generate homozygous BnPDAT1 overexpression canola lines, (2) generate homozygous PDAT1 knock-out canola lines using CRISPR/Cas9 technology, (3) evaluate responses to heat and drought stresses in canola lines with PDAT modification, and (4) conduct transcriptomic analysis on selected interesting lines to identify novel candidate genes.

Homozygous lines overexpressing the BnPDAT1 gene under the constitutive 35S promoter (BnDGAT1-OE) were successfully generated. BnPDAT1 knockout (BnPDAT1-KO) lines were developed using CRISPR/Cas9 technology, but even with great efforts, no true knockout lines were identified. As an alternative approach, BnPDAT1 knockdown (BnPDAT1-KD) lines were then developed with RNAi technology. The results of the stress treatments indicated that BnPDAT1-OE canola lines performed significantly better when exposed to heat stress or combined heat and drought stress at the reproductive stage, but not under drought stress along, as evidenced by the production of more pods per plant, seeds per pod, and yield per plant, indicating their promising value for canola breeding. Also interestingly, compared with the wild-type canola control, the BnDGAT1-OE lines showed a trend of higher total lipid content under both heat and combined heat and drought stress, and significantly higher oleic acid content under combined heat and drought stress. Subsequent transcriptomic analysis of the BnPDAT1-OE lines identified a number of novel candidate genes that were significantly altered under heat stress and combined heat and drought stress, which can be further characterized and used to improve abiotic stress tolerance in canola. The results can also be used to explore the molecular mechanism by which PDAT1 enhances canola’s tolerance to the abiotic stress. In addition to the originally proposed research objective, we also tested the BnPDAT1-OE canola lines at the seedling stage to tackle if the canola lines have better performance. The preliminary results indicated that PDAT1-OE might have better performance under cold or drought stress at the seedling stage, which are worth of validation and further study.

In conclusion, our results revealed that upregulation of PDAT can effectively improve canola’s performance under heat stress and combined heat and drought stress at the reproductive stage and identified a number of candidate genes with the potential to further improve abiotic stress tolerance in canola. The results also indicated that it would be interesting to comprehensively test the homozygous canola lines at the seedling, vegetative and flowering/seed development stages, with the aim of fully evaluating the potential of using BnPDAT1 and the associated canola lines in breeding canola for heat and drought tolerance. Moreover, it would be attractive to finely regulate PDAT to generate canola lines with good performance in both normal and non-ideal years. In addition, the results suggested that it would be valuable to evaluate the PDAT gene expression in a genetically diverse canola population to identify those with improved heat and drought tolerance and naturally high PDAT expression, and to incorporate the canola germplasm in canola breeding.