The FW2.2-like (FWL) genes encode cysteine-rich proteins with a placenta-specific 8 domain. They play roles in cell division and organ size control, response to rhizobium infection, and metal ion homeostasis in plants. Here, we target eight rice FWL genes using the CRISPR/Cas9 system delivered by Agrobacterium-mediated transformation. We successfully generate transgenic T₀ lines for 15 of the 16 targets. The targeted mutations are detected in the T₀ lines of all 15 targets and the average mutation rate is found to be 81.6%. Transfer DNA (T-DNA) truncation is a major reason for the failure of mutagenesis in T₀ plants. T-DNA segregation analysis reveals that the T-DNA inserts in transgenic plants can be easily eliminated in the T₁ generation. Of the 30 putative off-target sites examined, unintended mutations are detected in 13 sites. Phenotypic analysis reveals that tiller number and plant yield of OsFWL4 gene mutants are significantly greater than those of the wild type. Flag leaves of OsFWL4 gene mutants are wider than those of the wild type. The increase in leaf width of the mutants is caused by an increase in cell number. Additionally, grain length of OsFWL1 gene mutants is higher than that of the wild type. Our results suggest that transgene-free rice plants with targeted mutations can be produced in the T₁ generation using the Agrobacterium-mediated CRISPR/Cas9 system and that the OsFWL4 gene is a negative regulator of tiller number and plant yield.

Micronutrient metals, such as Mn, Cu, Fe, and Zn, are essential heavy metals for plant growth and development, while Cd is a nonessential heavy metal that is highly toxic to both plants and humans. Our understanding of the molecular mechanisms underlying Cd and micronutrient metal accumulation in plants remains incomplete. Here, we show that OsFWL7, an FW2.2-like (FWL) family gene in Oryza sativa, is preferentially expressed in the root and encodes a protein localized to the cell membrane. The osfwl7 mutation reduces both the uptake and the root-to-shoot translocation of Cd in rice plants. Additionally, the accumulation of micronutrient metals, including Mn, Cu, and Fe, was lower in osfwl7 mutants than in the wildtype plants under normal growth conditions. Moreover, the osfwl7 mutation affects the expression of several heavy metal transporter genes. Protein interaction analyses reveal that rice FWL proteins interact with themselves and one another, and with several membrane microdomain marker proteins. Our results suggest that OsFWL7 is involved in Cd and micronutrient metal accumulation in rice. Additionally, rice FWL proteins may form oligomers and some of them may be located in membrane microdomains.

Micronutrient deficiencies adversely affect human health and pose a significant global threat. Enhancing the accumulation of micronutrients in the edible parts of crops through genetic breeding is a promising strategy to mitigate micronutrient deficiencies in humans. FW2.2-like (FWL) genes play crucial roles in regulating heavy metal homeostasis in plants. We previously obtained two allelic mutants for each of the rice OsFWL1 (osfwl1a and osfwl1b) and OsFWL2 (osfwl2a and osfwl2b) genes. In this study, we showed that disruption of either OsFWL1 or OsFWL2 significantly enhanced the accumulation of metallic micronutrients in brown rice. Compared with that in the wild type, the iron (Fe) concentration in brown rice was higher in the osfwl1a (+166.7%), osfwl1b (+24.3%), and osfwl2a (+99.2%) mutants; the manganese (Mn) concentration was elevated in all four mutants (+25.1% to 35.6%); the copper (Cu) concentration increased in osfwl2a (+31.0%) and osfwl2b (+29.0%); and the zinc (Zn) concentration increased in osfwl2a (+10.2%). Additionally, disruption of OsFWL1 or OsFWL2 affected the homeostasis of metallic micronutrients in seedlings. Transcriptome analysis suggested that OsFWL1 and OsFWL2 might regulate cell wall polysaccharide metabolism and the expression of heavy metal transporter genes. Protein interaction analysis revealed that OsFWL1 interacted with OsFWL2 on the cell membrane. These findings suggest that OsFWL1 and OsFWL2 can serve as genetic biofortification tools to increase the concentrations of metallic micronutrients in rice grains.