Profiling of thylakoid complexes from the mesophyll and bundle sheath cells of C4, C3 and C2 Panicum grasses by BN/page and LC/MS

Hernandez-Prieto MA1, Foster C2, Watson-Lazowski A2, Ghannoum O2 and Chen M1

  1. ARC Centre of Excellence for Translational Photosynthesis, The University of Sydney, School of Life and Environmental Sciences, University of Sydney, NSW 2006. Australia.
  2. ARC Centre of Excellence for Translational Photosynthesis, Hawkesbury Institute for the Environment, Western Sydney University, Locked Bag 1797, Penrith NSW 2751, Australia.

Photosynthetic efficiency is largely limited because of the low specificity of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). C4 plants have higher efficiency compared to C3 plants because they use two carboxylation cycles, physically separated into two cell types: the mesophyll (MC) and the bundle sheath cells (BSC). This permits C4 plants to concentrate CO2 around RuBisCO, which favors the carboxylation pathway over oxygenation. The fact that C4 photosynthesis evolved independently more than 60 times (Osborne and Sack, 2012) is indicative of its success. Depending on the enzyme responsible for C4 decarboxylation, C4 plants can be divided into different biochemical subtypes: NADP-malic enzyme (ME), NAD-ME, and phosphoenolpyruvate carboxykinase (PEP-CK). The plant genus Panicum, similarly to Flaveria, contains species that perform typical C3 photosynthesis (P. bisulcatum), C2 intermediates (P. milioides), as well as representative of the main C4 biochemical subtypes: C4/NADP-ME (P. antidotale), C4/NAD-ME (P. miliaceum), and C4/PCK (P. maximumrenamed Megathyrsus maximus). Because of the interest in introducing some of the characteristic of C4 photosynthesis into C3 crops, the study of plant families representing the evolutionary and photosynthetic gradient from C3 to C4 is required. An important aspect of this transition that needs to be compared is the organization of the complexes involved in the light reactions to compensate for the higher demand of ATP in C4 plants. To compare the chloroplast organization of these species, we tested different methods to separate BSC chloroplasts from MC chloroplasts, and optimized the thylakoid preparation to obtain comparable band patterning in Native polyacrylamide gel electrophoresis (PAGE) under non-denaturing (native) conditions. Our results, comprising the analysis of 130 bands, showed that both C4 plants of the NADP-ME subtype analyzed have a higher proportion of PSI and NDH complexes with respect to PSII in BSC than in MC. No such imbalance was observed in C4 species of the other biochemical subtypes.