Comparative analysis of thylakoid protein complexes in the mesophyll and bundle sheath cells from C3 , C4 and C3 -C4 Paniceae grasses.

To better understand the coordination between dark and light reactions during the transition from C3 to C4 photosynthesis, we optimized a method for separating thylakoids from mesophyll (MC) and bundle sheath cells (BSCs) across different plant species. We grew six Paniceae grasses including representatives from the C3 , C3 -C4 and C4 photosynthetic types and all three C4 biochemical subtypes [nicotinamide adenine dinucleotide phosphate-dependent malic enzyme (NADP-ME), nicotinamide adenine dinucleotide-dependent malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK)] in addition to Zea mays under control conditions (1000 µmol quanta m-2  s-1 and 400 ppm of CO2 ). Proteomics analysis of thylakoids under native conditions, using blue native polyacrylamide gel electrophoresis followed by liquid chromatography-mass spectrometry (LC-MS), demonstrated the presence of subunits of all light-reaction-related complexes in all species and cell types. C4 NADP-ME species showed a higher photosystems I/II ratio and a clear accumulation of the NADH dehydrogenase-like complexes in BSCs, while Cytb6 f was more abundant in BSCs of C4 NAD-ME species. The C4 PEPCK species showed no clear differences between cell types. Our study presents, for the first time, a good separation between BSC and MC for a C3 -C4 intermediate grass which did not show noticeable differences in the distribution of the thylakoid complexes. For the NADP-ME species Panicum antidotale, growth at glacial CO2 (180 ppm of CO2 ) had no effect on the distribution of the light-reaction complexes, while growth at low light (200 µmol quanta m-2  s-1 ) promoted the accumulation of light-harvesting proteins in both cell types. These results add to our understanding of thylakoid distribution across photosynthetic types and subtypes, and introduce thylakoid distribution between the MC and BSC of a C3 -C4 intermediate species.