Genetic modification of flavone biosynthesis in rice enhances biofilm formation of soil diazotrophic bacteria and biological nitrogen fixation

Abstract

Improving biological nitrogen fixation (BNF) in cereal crops is a long-sought objective; however, no successful modification of cereal crops showing increased BNF has been reported. Here, we described a novel approach in which rice plants were modified to increase the production of compounds that stimulated biofilm formation in soil diazotrophic bacteria, promoted bacterial colonization of plant tissues and improved BNF with increased grain yield at limiting soil nitrogen contents. We first used a chemical screening to identify plant-produced compounds that induced biofilm formation in nitrogen-fixing bacteria and demonstrated that apigenin and other flavones induced BNF. We then used CRISPR-based gene editing targeting apigenin breakdown in rice, increasing plant apigenin contents and apigenin root exudation. When grown at limiting soil nitrogen conditions, modified rice plants displayed increased grain yield. Biofilm production also modified the root microbiome structure, favouring the enrichment of diazotrophic bacteria recruitment. Our results support the manipulation of the flavone biosynthetic pathway as a feasible strategy for the induction of biological nitrogen fixation in cereals and a reduction in the use of inorganic nitrogen fertilizers.

The Impact of Non-Nodulating Diazotrophic Bacteria in Agriculture: Understanding the Molecular Mechanisms That Benefit Crops

“…Similar investigations have been performed in maize [ 50 , 52 , 67 , 68 , 69 ], rice [ 47 , 70 , 71 ], wheat [ 72 ], mung bean ( Vigna radiata ) [ 17 ], cowpea bean ( Vigna unguiculata L. Walp) [ 53 , 73 ], juçara palm ( Euterpe edulis Mart.) [ 18 ], and tomato ( Solanum lycopersicum ) [ 19 ].…”

“…In addition to plant genotype, several studies suggest that there is a decisive refinement of the bacterial community in the rhizoplane in close contact with the plant host on the root surface and at the emergence points of the lateral roots [ 38 , 68 , 70 , 74 ]. Biofilm formation or specific adhesion mechanisms may be key factors in this enrichment step, essentially modifying the root microbiome structure.…”

“…Biofilm formation or specific adhesion mechanisms may be key factors in this enrichment step, essentially modifying the root microbiome structure. In a novel study, rice plants engineered with CRISPR/Cas9 to modify a flavone biosynthetic pathway generated apigenin-enriched rice plants that extruded apigenin into the rhizosphere [ 70 ]. Consequently, the increased production of this flavone stimulated biofilm formation in diazotrophic soil bacteria, improving the colonization of diazotrophic bacteria in rice plant tissues and promoting BNF [ 70 ].…”

“…In a novel study, rice plants engineered with CRISPR/Cas9 to modify a flavone biosynthetic pathway generated apigenin-enriched rice plants that extruded apigenin into the rhizosphere [ 70 ]. Consequently, the increased production of this flavone stimulated biofilm formation in diazotrophic soil bacteria, improving the colonization of diazotrophic bacteria in rice plant tissues and promoting BNF [ 70 ]. Interestingly, a recent study carried out in nitrogen-depleted fields in Oaxaca, Mexico demonstrated that maize plants have developed an extensive network of mucilage-secreting aerial roots that harbor a microbiome rich in N 2 -fixing species [ 68 ].…”

Deciphering the potential of a plant growth promoting endophyte Rhizobium sp. WYJ-E13, and functional annotation of the genes involved in the metabolic pathway

“…Furthermore, motility and chemotaxis were important characteristics for endophytes ( Taghavi et al, 2010 ). Recently, the biofilm formation of diazotrophic bacteria Gluconacetobacter diazotrophicus was associated with the stimulation of certain compounds, and promoted its motility and colonization into the host plant, thus further improving biological nitrogen fixation with increased grain yield ( Yan et al, 2022 ). In WYJ-E13 genome, a total of 18 genes were associated with motility ( Table 4 ).…”