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 Cytb₆f 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.