Improved analysis of C4 and C3 photosynthesis via refined in vitro assays of their carbon fixation biochemistry.
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, Mg(2+), 22 °C) and phosphoenolpyruvate carboxykinase (pH 7, >2mM Mn(2+), no Mg(2+)) 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.
ARC Centre of Excellence for Translational Photosynthesis, Research School of Biology, Australian National University, Canberra ACT 2601, Australia robert.sharwood@anu.edu.au.
Robert E
RE
Sharwood
ARC Centre of Excellence for Translational Photosynthesis, Hawkesbury Institute for the Environment, Western Sydney University, Richmond NSW 2753, Australia.
Balasaheb V
BV
Sonawane
ARC Centre of Excellence for Translational Photosynthesis, Hawkesbury Institute for the Environment, Western Sydney University, Richmond NSW 2753, Australia.
Oula
O
Ghannoum
ARC Centre of Excellence for Translational Photosynthesis, Research School of Biology, Australian National University, Canberra ACT 2601, Australia.
Spencer M
SM
Whitney
D002245:Carbon Dioxide / Q000378:metabolism; D066298:In Vitro Techniques; D050538:Malate Dehydrogenase (NADP+) / Q000378:metabolism; D008897:Panicum / Q000378:metabolism; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000378:metabolism; D010788:Photosynthesis / Q000502:physiology*; D018515:Plant Leaves / Q000201:enzymology / Q000378:metabolism; D006109:Poaceae / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D014026:Nicotiana / Q000378:metabolism; D014908:Triticum / Q000378:metabolism; D003313:Zea mays / Q000378:metabolism
D002245:Carbon Dioxide; D050538:Malate Dehydrogenase (NADP+); D012273:Ribulose-Bisphosphate Carboxylase; D019764:Phosphoenolpyruvate Carboxykinase (ATP)
CO2-concentrating mechanism; Carbamylation; Rubisco; Rubisco activase.; carbon fixation; photosynthesis