Improved analysis of C(4) and C(3) photosynthesis via refined in vitro assays of their carbon fixation biochemistry

Plants operating C(3) and C(4) 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 C(3) and C(4) 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, Mg(2+), 22 °C) and phosphoenolpyruvate carboxykinase (pH 7, >2mM Mn(2+), no Mg(2+)) maximal activities accurately. We validate how the omission of MgCl(2) 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 C(4) monocot leaves (45–60% activation) relative to C(3) monocots (55–90% activation). Consistent with their >3-fold lower Rubisco contents, full Rubisco activation in soluble protein from C(4) leaves (<5min) was faster than in C(3) 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.