The ferredoxin/thioredoxin pathway constitutes an indispensable redox-signaling cascade for light-dependent reduction of chloroplast stromal proteins

To ensure efficient photosynthesis, chloroplast proteins need to be flexibly regulated under fluctuating light conditions. Thiol-based redox regulation plays a key role in reductively activating several chloroplast proteins in a light-dependent manner. The ferredoxin (Fd)/thioredoxin (Trx) pathway has long been recognized as the machinery that transfers reducing power generated by photosynthetic electron transport re-actions to redox-sensitive target proteins; however, its biolog-ical importance remains unclear, because the complete disruption of the Fd/Trx pathway in plants has been unsuc-cessful to date. Especially, recent identifications of multiple redox-related factors in chloroplasts, as represented by the NADPH-Trx reductase C, have raised a controversial proposal that other redox pathways work redundantly with the Fd/Trx pathway. To address these issues directly, we used CRISPR/ Cas9 gene editing to create Arabidopsis mutant plants in which the activity of the Fd/Trx pathway was completely defective. The mutants generated showed severe growth inhibition. Importantly, these mutants almost entirely lost the ability to reduce several redox-sensitive proteins in chloroplast stroma, including four Calvin-Benson cycle enzymes, NADP-malate dehydrogenase, and Rubisco activase, under light conditions. These striking phenotypes were further accompanied by abnormally developed chloroplasts and a drastic decline in photosynthetic efficiency. These results indicate that the Fd/ Trx pathway is indispensable for the light-responsive activation of diverse stromal proteins and photoautotrophic growth of plants. Our data also suggest that the ATP synthase is excep-tionally reduced by other pathways in a redundant manner. This study provides an important insight into how the chlo-roplast redox-regulatory system operates in vivo.

Automated summary

To ensure efficient photosynthesis, the regulation of chloroplast proteins under changing light conditions is crucial. This study investigates the biological importance of the ferredoxin/thioredoxin pathway in activating chloroplast proteins. By using gene editing technology, Arabidopsis mutant plants with a completely defective ferredoxin/thioredoxin pathway were created, resulting in severe growth inhibition and loss of ability to reduce several redox-sensitive proteins in chloroplast stroma. The study provides important insights into the operation of the chloroplast redox-regulatory system in vivo.