C4Grasses Employ Distinct Strategies to Acclimate Rubisco Activase to Heat Stress

1 Rising temperatures due to the current climate crisis will have devastating impacts on crop performance and resilience in the near future. One key step that limits plant photosynthetic performance under higher temperatures is the activity of the thermolabile enzyme rubisco activase (RCA). RCA is highly conserved in photosynthetic organisms, including C 4 crops such as Zea mays (maize) and Sorghum bicolor (sorghum) which are crucial components of global food supply and the bioenergy sector. While rubisco is the most abundant protein on earth and responsible for carbon fixation, RCA is an essential chaperone required to remove inhibitory sugar phosphates from the active site of rubisco to allow for continued CO 2 fixation. We set out to understand temperature-dependent RCA regulation in four different C 4 plants, with a focus on the crop plants maize (two cultivars) and sorghum, as well as the model grass Setaria viridis (setaria). Gas exchange measurements confirm that CO 2 assimilation is indeed limited by Ribulose 1,5-bisphosphate (RuBP) carboxylation in these organisms and at high temperatures. All three species express distinct sets of RCA isoforms and each species alters the isoform and proteoform abundances in response to heat; however, the changes are species-specific. In order to understand how even subtle changes in the molecular environment of the chloroplast stroma affect RCA function during heat acclimation, we examined the regulation of RCA activity directly with respect thermostability, the ratio of ADP to ATP and the concentration of Mg 2+ ions. As shown previously, the activity of RCA is modulated by a combination of these variables, but surprisingly, how these biochemical environment factors affect RCA function differs vastly between the different C 4 species, and differences are even apparent between different cultivars within a single species, both with respect to proteoform abundance and regulation. Our results suggest that each grass evolved different parts of the RCA regulation portfolio and we conclude that a successful engineering approach aimed at improving carbon capture in C4 grasses will need to accommodate these individual regulatory mechanisms.