Spectrophotometric Determination of RuBisCO Activity and Activation State in Leaf Extracts
RuBisCO plays a central role in photosynthesis and, due to its catalytic inefficiencies, frequently limits CO2 assimilation in fully illuminated leaves at the top of unstressed crop canopies. The CO2-fixing enzyme is heavily regulated and not all the enzyme present in the leaf is active at any given moment. In this chapter, a spectrophotometric assay is described for measuring RuBisCO activity and activation state in leaf extracts. Most of the assay components are available commercially and others can be produced by established protocols, making adoption of the assay achievable by most plant biochemistry laboratories. Its relative high-throughput capacity enables large-scale experiments aimed at screening germplasm for improved RuBisCO function.
Key wordsCarboxylation Enzyme activity Activation state Spectrophotometry Microplate reader NADH PK-LDH 14CO2
We thank Dr. Mike Salvucci (previously USDA-ARS) for useful discussions, Prof. Rebekka Wachter and Mr. Matthew Hilton (Arizona State University) for advice on implementing this method in our laboratory, and Dr. Doug Orr for helpful comments on the manuscript. CRGS and ECS acknowledge funding from the International Wheat Yield Partnership (IWYP64). ECS also acknowledges support from a sub-contract to the Bill & Melinda Gates Foundation award RIPE, Realizing Increased Photosynthetic Efficiency.
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