Engineering Enhanced Photosynthesis in Zea mays

Zea mays (Maize, Zm) and Miscanthus x giganteus (Mg) employ C4 photosynthesis, which enhances carbon assimilation efficiency by concentrating CO2 around Rubisco to minimize photorespiration. Pyruvate, orthophosphate dikinase (PPDK) regenerates phosphoenolpyruvate from pyruvate to feed the carbon concentrating cycle. PPDK is regulated by PPDK regulatory protein (PDRP) through phosphorylation and dephosphorylation. PPDK is a tetramer, and in maize the tetramer dissociates between 10-15°C, leading to decreased photosynthesis under chilling conditions. Conversely, M. x giganteus is chilling tolerant and maintains PPDK abundance and activity. This study investigates the impacts of expressing MgPPDK in maize alongside the endogenous PPDK and alone in PPDK mutants. Homozygous maize PPDK mutants (ΔZm) are seedling-lethal, but plants expressing the MgPPDK transgene without the endogenous ZmPPDK (MgΔZm) are viable, demonstrating successful complementation. Three genotypes were analyzed in this study; wild-type, azygous segregants of transgenic lines ; MgΔZm; and +Mg, where plants contain both ZmPPDK and MgPPDK. Plants were grown under high and low light and genotyped using PCR and immunoblots. Leaf protein samples from several time points were analyzed using immunoblots, screening for phosphorylated (inactive) PPDK, total PPDK content, transgenic PPDK content, and total protein levels. Carbon assimilation was measured using a LI-6800 instrument at several points during the day-night cycle. From this study, we determined the impact that MgPPDK has on protein content and photosynthetic performance in maize. MgΔZm plants had higher transgene expression than +Mg and lower inactive PPDK than the other genotypes. However, they had poor photosynthetic performance in low light. In maize, MgPPDK may have better substrate interactions with endogenous PDRP than ZmPPDK does, but weaker tetramer associations. Gaining a better understanding of these plants will lay the groundwork for future experiments to explore the effects of higher transgene expression and observe these plants under different environmental conditions.

Working with Dr. Stern’s lab under Riley’s mentorship this summer has been an incredible experience, and I am excited to employ my newfound tools and skills in future research efforts. I have been researching photosynthesis with the RIPE project at the University of Illinois for 2 years, but this was my first time playing a role in the real-world applications of formal academia. I am grateful for the independence that I was entrusted with while being supported by a collaborative and encouraging lab group. Many things went wrong over the course of this program, including changing my project entirely several weeks into the summer, but I am grateful for the opportunity to challenge myself and problem-solve. I will miss the community-driven culture of the Stern lab, and I am eager to carry this experience to wherever a career in agricultural engineering leads me.