Increasing yield potential through manipulating of an ARE1 ortholog related to nitrogen use efficiency in wheat by CRISPR/Cas9

Wheat (Triticum aestivum L.) is a staple food crop consumed by more than 30% of world population. Nitrogen (N) fertilizer has been applied broadly in agriculture practice to improve wheat yield to meet the growing demands for food production. However, undue N fertilizer application and the low N use efficiency (NUE) of modern wheat varieties are aggravating environmental pollution and ecological deterioration. Under nitrogen-limiting conditions, the rice (Oryza sativa) abnormal cytokinin response1 repressor1 (are1) mutant exhibits increased NUE, delayed senescence and consequently, increased grain yield. However, the function of ARE1 ortholog in wheat remains unknown. Here, we isolated and characterized three TaARE1 homoeologs from the elite Chinese winter wheat cultivar ZhengMai 7698. We then used CRISPR/Cas9-mediated targeted mutagenesis to generate a series of transgene-free mutant lines either with partial or triple-null taare1 alleles. All transgene-free mutant lines showed enhanced tolerance to N starvation, and showed delayed senescence and increased grain yield in field conditions. In particular, the AABBdd and aabbDD mutant lines exhibited delayed senescence and significantly increased grain yield without growth defects compared to the wild-type control. Together, our results underscore the potential to manipulate ARE1 orthologs through gene editing for breeding of high-yield wheat as well as other cereal crops with improved NUE.

Automated summary

Wheat is a staple food consumed by a large portion of the global population, with nitrogen fertilizer being widely used to increase yield. However, excessive nitrogen use and low nitrogen use efficiency of modern wheat varieties have led to environmental pollution. This study identified three TaARE1 homoeologs from elite Chinese winter wheat cultivars and used CRISPR/Cas9 technology to generate transgene-free mutant wheat lines with enhanced nitrogen starvation tolerance, delayed senescence, and increased grain yield. The results highlight the potential of manipulating ARE1 orthologs through gene editing to breed high-yield wheat and other cereal crops with improved nitrogen use efficiency.