Towards an integrative model of C4 photosynthetic subtypes: insights from comparative transcriptome analysis of NAD-ME, NADP-ME, and PEP-CK C4 species

C-4 photosynthesis affords higher photosynthetic carbon conversion efficiency than C-3 photosynthesis and it therefore represents an attractive target for engineering efforts aiming to improve crop productivity. To this end, blueprints are required that reflect C-4 metabolism as closely as possible. Such blueprints have been derived from comparative transcriptome analyses of C-3 species with related C-4 species belonging to the NAD-malic enzyme (NAD-ME) and NADP-ME subgroups of C-4 photosynthesis. However, a comparison between C-3 and the phosphoenolpyruvate carboxykinase (PEP-CK) subtype of C-4 photosynthesis is still missing. An integrative analysis of all three C-4 subtypes has also not been possible to date, since no comparison has been available for closely related C-3 and PEP-CK C-4 species. To generate the data, the guinea grass Megathyrsus maximus, which represents a PEP-CK species, was analysed in comparison with a closely related C-3 sister species, Dichanthelium clandestinum, and with publicly available sets of RNA-Seq data from C-4 species belonging to the NAD-ME and NADP-ME subgroups. The data indicate that the core C-4 cycle of the PEP-CK grass M. maximus is quite similar to that of NAD-ME species with only a few exceptions, such as the subcellular location of transfer acid production and the degree and pattern of up-regulation of genes encoding C-4 enzymes. One additional mitochondrial transporter protein was associated with the core cycle. The broad comparison identified sucrose and starch synthesis, as well as the prevention of leakage of C-4 cycle intermediates to other metabolic pathways, as critical components of C-4 metabolism. Estimation of intercellular transport fluxes indicated that flux between cells is increased by at least two orders of magnitude in C-4 species compared with C-3 species. In contrast to NAD-ME and NADP-ME species, the transcription of photosynthetic electron transfer proteins was unchanged in PEP-CK. In summary, the PEP-CK blueprint of M. maximus appears to be simpler than those of NAD-ME and NADP-ME plants.