Functional relocation of the maize chloroplast atpB gene to the nucleus restores photosynthetic competence to a gene-edited non-photosynthetic mutant

With the increasing food demands of a global population projected to reach 9.6 billion by 2050, there is an urgent need to increase crop productivity. Bioengineering approaches to boost crop yields include enhancing photosynthetic capacity, though relatively few efforts focus on C4 crop species despite their significant presence in agriculture. Multiple photosynthesis engineering examples utilize overexpression of components of the nuclear-encoded machinery, while work on chloroplast-encoded photosynthetic genes is limited to the few dicot species where plastid transformation technology exists. We present here a novel approach to photosynthetic gene engineering in maize using a nuclear-encoded, chloroplast-targeted TALE-cytidine deaminase enzyme to create non-photosynthetic knockout mutants of the chloroplast rbcL gene. An off-target mutation in the adjacent atpB gene, encoding the β subunit of ATP synthase, was consistently found in all edited lines, identified as pigment-deficient in tissue culture. These double mutants, carrying mutations in both genes, were purified to homoplasmy using unique leaf-base regeneration techniques. To test mutation complementation and identify the causal gene, nuclear transgenic lines overexpressing chloroplast-targeted RbcL and AtpB proteins were generated. The results show that nuclear expression of AtpB restores chlorophyll accumulation and supports wild-type growth in tissue culture. Nonphotochemical quenching (NPQ) function was restored, and the maximum quantum yield of photosystem II (Fv/Fm) reached about 30% of wild-type levels in the nuclear-transformed lines. This is the first demonstration in a monocot plant that complementation of a photosynthetic mutant via nuclear gene expression is possible, providing a facile method for future photosynthetic engineering.