Abstract
Nitrogen availability remains a principal constraint to crop productivity. Plants cannot directly assimilate the abundant nitrogen available in our atmosphere; instead, they rely on the uptake of inorganic forms of nitrogen, such as ammonium and nitrate from the soil. Nitrogen is a limiting nutrient in wheat production, and wheat yields are very responsive to nitrogen fertilisation. Only diazotrophic bacteria can convert atmospheric nitrogen to ammonia via biological nitrogen fixation (BNF), and although improving BNF in wheat has been a longstanding objective, there have been no descriptions of successful modification of wheat crops showing increased BNF in the literature. Here we describe the use of polycistronic multiplexed CRISPR to modify the flavone biosynthetic pathway of hexaploid wheat (Triticum aestivum) plants, generating DNA-edited plants with increased apigenin content. The apigenin-enriched plants exude apigenin into the soil, inducing the colonisation of the roots and subsequent formation of biofilms in soil by diazotrophic bacteria. The low permeability of the biofilm to oxygen protected the bacterial nitrogenase and stimulated BN. Under nitrogen-limiting conditions, apigenin-enriched wheat lines exhibited increased nitrogen content, improved photosynthetic performance, and higher grain yield relative to wild-type controls. This work demonstrates the feasibility of engineering associative BNF in cereals via metabolic reprogramming of root exudation, offering a sustainable route to reduce dependence on synthetic nitrogen fertilisers.