Towards an integrative model of C(4) photosynthetic subtypes: insights from comparative transcriptome analysis of NAD-ME, NADP-ME, and PEP-CK C(4) 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.