A point mutation in the gene encoding Mg-chelatase subunit I influences strawberry leaf color and metabolism.

Magnesium chelatase catalyzes the insertion of magnesium into protoporphyrin IX, a vital step in chlorophyll biogenesis. The enzyme consists of three subunits, (Magnesium chelatase I subunit, CHLI), (Magnesium chelatase D subunit, CHLD) and (Magnesium chelatase H subunit, CHLH). The CHLI subunit is an ATPase that mediates catalysis. Previous studies on CHLI have mainly focused on model plant species, and its functions in other species have not been well described, especially with regard to leaf coloration and metabolism. In this study, we identified and characterized a CHLI mutant in strawberry species Fragaria pentaphylla. The mutant, noted as p240, exhibits yellow-green leaves and a low chlorophyll (Chl) level. RNA-seq identified a mutation in the 186th amino acid of the CHLI subunit, a base conserved in most photosynthetic organisms. Transient transformation of wild-type CHLI into p240 leaves complemented the mutant phenotype. Further mutants generated from RNA-interference (RNAi) and CRISPR/Cas9 gene editing recapitulated the mutant phenotype. Notably, heterozygous chli mutants accumulated more chlorophyll under low light conditions compared to high light conditions. Metabolite analysis of null mutants under high light conditions revealed substantial changes in both nitrogen and carbon metabolism. Further analysis indicated that mutation in Glu186 of CHLI does not affect its subcellular localization, nor the interaction between CHLI and CHLD. However, intramolecular interactions were impaired, leading to reduced ATPase and magnesium chelatase activity. These findings demonstrate that Glu186 plays a key role in enzyme function, affecting leaf coloration via the formation of the hexameric ring itself, and that manipulation of CHLI may be a means to improve strawberry plant fitness and photosynthetic efficiency under low light conditions.