C4 Grasses Employ Various Strategies to Acclimate Rubisco Activase to Heat Stress

Abstract C 4 crops such as Zea mays (maize) and Sorghum bicolor (sorghum) are crucial producers of food and bioenergy. In these crops, carbon assimilation is limited at higher temperatures because the enzyme rubisco activase (RCA), which is responsible for proper function of the CO 2 fixing protein rubisco, has been shown to be thermolabile. We present a holistic approach to understand carbon assimilation and RCA function in maize, sorghum, and the C 4 model grass Setaria viridis (setaria) during heat stress acclimation. Gas exchange data confirms that assimilation is limited by Ribulose 1,5-bisphosphate (RuBP) carboxylation during heat. Plants express a variety of RCA isoforms and we show that each species changes the isoform expression and proteoform abundances of these variants in different ways. In addition, to understand how changing conditions in the chloroplast stroma affect RCA function during heat, we examined the regulation of RCA activity by thioredoxin F, magnesium ions and adenosine diphosphate. As expected, 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 species. Unexpected differences are even apparent within a single species: different cultivars of maize show differences in assimilation, proteoform abundance and regulation. Our surprising observation that each grass expresses and regulates RCA differently suggests that even closely related plants use different strategies to maintain RCA function and we conclude that a single reference system cannot accurately reflect protein function in every cultivar.