Comparative proteomics of chloroplast envelopes from C3 and C4 plants reveals specific adaptations of the plastid envelope to C4 photosynthesis and candidate proteins required for maintaining C4 metabolite fluxes

C-4 plants have up to 10-fold higher apparent CO2 assimilation rates than the most productive C-3 plants. This requires higher fluxes of metabolic intermediates across the chloroplast envelope membranes of C-4 plants in comparison with those of C-3 plants. In particular, the fluxes of metabolites involved in the biochemical inorganic carbon pump of C-4 plants, such as malate, pyruvate, oxaloacetate, and phosphoenolpyruvate, must be considerably higher in C-4 plants because they exceed the apparent rate of photosynthetic CO2 assimilation, whereas they represent relatively minor fluxes in C-3 plants. While the enzymatic steps involved in the C-4 biochemical inorganic carbon pump have been studied in much detail, little is known about the metabolite transporters in the envelope membranes of C-4 chloroplasts. In this study, we used comparative proteomics of chloroplast envelope membranes from the C-3 plant pea (Pisum sativum) and mesophyll cell chloroplast envelopes from the C-4 plant maize (Zea mays) to analyze the adaptation of the mesophyll cell chloroplast envelope proteome to the requirements of C-4 photosynthesis. We show that C-3- and C-4- type chloroplasts have qualitatively similar but quantitatively very different chloroplast envelope membrane proteomes. In particular, translocators involved in the transport of triosephosphate and phosphoenolpyruvate as well as two outer envelope porins are much more abundant in C-4 plants. Several putative transport proteins have been identified that are highly abundant in C-4 plants but relatively minor in C-3 envelopes. These represent prime candidates for the transport of C-4 photosynthetic intermediates, such as pyruvate, oxaloacetate, and malate.