Elucidating the role of PGR5 and PGRL1 in the regulation of C4 photosynthesis
Regulation of C4 photosynthesis is an elaborate process that requires cell-specific specialisation of the light reactions to match the distinct metabolic energy demands of mesophyll and bundle sheath cells. This balance is further complicated under fluctuating light conditions which require rapid changes in energy production and photoprotection, yet how C4 plants achieve this has remained poorly defined. PROTON GRADIENT REGULATION 5 (PGR5) and PGR5-LIKE 1 (PGRL1), which keep Photosystem I (PSI) oxidised by regulating PSI photoprotection and proton motive force generation, stood out as the principal candidates, accumulating to higher levels in C4 species than in their C3 relatives. This thesis provides novel insights into the evolution and function of the PGR5/PGRL1 system in the C4 model grass S. viridis, showing that it is not simply inherited from C3 plants but has specialised for the regulatory needs of C4 photosynthesis.
Phylogenetic analysis revealed that grasses carry two PGRL1 paralogs that diverged following a whole-genome duplication early in grass evolution: the ancestral PGRL1α, conserved across algae and land plants, and a grass-specific PGRL1β. In NADP-ME C4 grasses these paralogs are expressed in a cell-specific manner, with PGRL1α enriched in mesophyll cells and PGRL1β in bundle sheath cells. Analysis of gene-edited Setaria viridis showed that PGRL1β was necessary for faster oxidation of PSI during dark-to-high-light transitions, whereas PGRL1α was required to keep PSI sufficiently oxidised at steady-state high light affording a more dynamic range of photoprotection than in non-grasses.
To address the long-debated mechanism of PGR5, a mass-action kinetic framework was developed to convert in vivo Dark Interval Relaxation Kinetics (DIRK)–NIR redox signals of P700, plastocyanin, and ferredoxin into electron fluxes. This approach confirmed that loss of PGR5 is associated not only with acceptor-side limitation at PSI but also with a substantial donor-side flux deficit at 1 second post low-to-high-light transition that cannot be explained by decreased PSII activity, a restriction at Cytochrome b6f, or depletion of PSI acceptor capacity, providing new in vivo evidence that suggests PGR5 may regulate cyclic electron flow without concluding that PGR5 definitively participates in a cyclic electron flow pathway.
In C3 plants, pgr5 and pgrl1 mutants are phenotypically identical, but whether this holds in C4 had not been tested. Unexpectedly, the Setaria viridis pgrl1αβ mutant retained ~6% of wild-type PGR5 under fluctuating light. In result, the mutant showed an intermediate growth and photosynthetic phenotype between wild-type and pgr5-1. Because PSI declined as severely as in pgr5-1, this residual pool was too low to protect PSI and result in the intermediate growth; instead, it was associated with improved metabolic acclimation via elevated C4 and photorespiration enzymes. PGR5 therefore contributes to the coordinated metabolic acclimation of C4 plants to fluctuating light, a role beyond its established function in PSI photoprotection.
Together, these findings refine the mechanistic understanding of how PGR5 and PGRL1 act in C4 photosynthesis and identify new directions and priority targets for engineering improved electron transfer in C4 crops under the fluctuating light of field environments.