Targeted knockout of barley Ycf54 demonstrates its essential function in the Mg-protoporphyrin IX monomethyl ester...

The Mg-protoporphyrin IX monomethyl ester cyclase is one of the 15 enzymes required for biosynthesis of chlorophyll in plants. The Ycf54 protein is a component associated with this enzyme and probably functions as a chaperone. Due to the lack of mutants in the Ycf54 gene, we targeted this gene in barley (Hordeum vulgare L.) by RNA-guided Cas9 genome editing to dissect its role in chlorophyll biosynthesis.

Two guide RNAs were designed and delivered via Agrobacterium-mediated transformation. We generated four homozygous mutant alleles harboring deletions of 1, 2, 6 and 27 base pairs. The 27 bp deletion mutant also had a silent C-to-G point mutation before the deletion and a CC-to-AG mutation following the deletion changing a serine residue to glutamate. The mutants exhibited three distinct phenotypes: completely yellow leaves, green leaves with yellow stripes, and fully green leaves. Yellow-leafed plants were classified as Xantha mutants, characterized by an absence of chlorophyll but presence of carotenoids. The striped phenotype represented chimeric individuals with severe mutations in meristematic tissues. Yellow mutants carried homozygous 1 or 2 bp deletions, both of which caused translational reading frame shifts and premature truncation of the Ycf54 protein, likely disrupting chlorophyll biosynthesis and resulting in lethality. These mutations could only be stored in heterozygous lines. In contrast, the 6 and 27 bp deletions were in-frame and did not have any observed effect on the green phenotype, demonstrating that these mutations, and the CC-to-AG mutation, preserve Ycf54 functionality with maintained chlorophyll synthesis and plant viability.

Previous studies have implicated Ycf54 in the folding and maturation of the cyclase enzyme. Our findings provide direct genetic evidence that Ycf54 is essential for chlorophyll biosynthesis. Loss-of-function mutations result in a lethal, chlorophyll-deficient phenotype, underscoring the critical role of Ycf54 in photosynthetic development.