Improved analysis of C₄ and C₃ photosynthesis via refined in vitro assays of their carbon fixation biochemistry

Abstract

Plants operating C₃ and C₄ photosynthetic pathways exhibit differences in leaf anatomy and photosynthetic carbon fixation biochemistry. Fully understanding this underpinning biochemical variation is requisite to identifying solutions for improving photosynthetic efficiency and growth. Here we refine assay methods for accurately measuring the carboxylase and decarboxylase activities in C₃ and C₄ plant soluble protein. We show that differences in plant extract preparation and assay conditions are required to measure NADP-malic enzyme and phosphoenolpyruvate carboxylase (pH 8, Mg²⁺, 22 °C) and phosphoenolpyruvate carboxykinase (pH 7, >2mM Mn²⁺, no Mg²⁺) maximal activities accurately. We validate how the omission of MgCl₂ during leaf protein extraction, lengthy (>1min) centrifugation times, and the use of non-pure ribulose-1,5-bisphosphate (RuBP) significantly underestimate Rubisco activation status. We show how Rubisco activation status varies with leaf ontogeny and is generally lower in mature C₄ monocot leaves (45-60% activation) relative to C₃ monocots (55-90% activation). Consistent with their >3-fold lower Rubisco contents, full Rubisco activation in soluble protein from C₄ leaves (<5min) was faster than in C₃ plant samples (<10min), with addition of Rubisco activase not required for full activation. We conclude that Rubisco inactivation in illuminated leaves primarily stems from RuBP binding to non-carbamylated enzyme, a state readily reversible by dilution during cellular protein extraction. Show more