Abstract - Cyclic electron flow (CEF) around PSI is essential for balancing ATP/NADPH supply and protecting photosystems under fluctuating light. In grasses, a duplication of PGRL1 gave rise to two paralogs, PGRL1α and PGRL1β which show differential expression in the two C4 cell types: Mesophyll and Bundle sheath. Although this cell enriched expression pattern was previously discovered, their functional divergence has remained unresolved. Using Setaria viridis CRISPR-cas9 mutants, I combined spectroscopic and physiological approaches to dissect their roles. For the first time, I directly quantified the rates of electron transfer into and out of PSI during PGR5/PGRL1-dependent CEF, revealing distinct contributions of the paralogs to photoprotection under high light. While PGRL1β enables rapid initiation of PSI oxidation during light transitions, loss of PGRL1α reduces PSI donor-side limitation at high irradiance, consistent with its role as the more abundant paralog. The pgrl1αβ double mutant revealed an unexpected phenotype: in contrast to Arabidopsis, where pgrl1ab is identical to pgr5, Setaria pgrl1αβ mutants were more resilient to fluctuating light than pgr5. Strikingly, although pgrl1αβ and pgr5 mutants both lacked similar amounts of CEF at high light, electrochromic shift measurements showed that only pgrl1αβ retained partial regulation of ATP synthase activity. This divergence suggests that residual PGR5 may be present in the background of pgrl1αβ , and therefore PGR5 has an additional role in modulating ATP synthase beyond its established role in PSI cyclic flow. Together, these findings establish functional partitioning of PGRL1 paralogs in C₄photosynthesis and uncover a new regulatory dimension of PGR5 participating in CEF and ATP synthase regulation.

Biography - Samuel “James” Nix completed his undergraduate studies in 2018 at Abilene Christian University (Abilene, Texas), where he majored in Biology, Biochemistry, and Chemistry. He then worked as a Food and Formulation Chemist at Next Level Nutraceuticals before moving to Australia in 2020. James completed a Master’s degree in Biotechnology in 2022 under the supervision of Robert Furbank, Maria Ermakova, and Marten Moore, focusing on photosynthetic physiology. He then continued on with a PhD in the Furbank Lab adding Kai Chan to his list of supervisors and was affiliated with the ARC Training Centre for Future Crops Development.

His doctoral research investigates the role of PGRL1 paralogs in cyclic electron flow of C₄ photosynthesis, combining CRISPR-based mutant generation, proteomics, phylogenetics, and advanced spectroscopic techniques such as Dual-KLAS-NIR. Alongside these approaches, he has also gained experience in protein modeling, synthetic biology, and qPCR-based analyses. His long-term research goal is to improve C₄ crop yields by optimizing electron transport and PSI protection under dynamic light environments.