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

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.