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

Abstract

C4 photosynthesis affords higher photosynthetic carbon conversion efficiency than C3 photosynthesis and it therefore represents an attractive target for engineering efforts aiming to improve crop productivity. To this end, blueprints are required that reflect C4 metabolism as closely as possible. Such blueprints have been derived from comparative transcriptome analyses of C3 species with related C4 species belonging to the NAD-malic enzyme (NAD-ME) and NADP-ME subgroups of C4 photosynthesis. However, a comparison between C3 and the phosphoenolpyruvate carboxykinase (PEP-CK) subtype of C4 photosynthesis is still missing. An integrative analysis of all three C4 subtypes has also not been possible to date, since no comparison has been available for closely related C3 and PEP-CK C4 species. To generate the data, the guinea grass Megathyrsus maximus, which represents a PEP-CK species, was analysed in comparison with a closely related C3 sister species, Dichanthelium clandestinum, and with publicly available sets of RNA-Seq data from C4 species belonging to the NAD-ME and NADP-ME subgroups. The data indicate that the core C4 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 C4 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 C4 cycle intermediates to other metabolic pathways, as critical components of C4 metabolism. Estimation of intercellular transport fluxes indicated that flux between cells is increased by at least two orders of magnitude in C4 species compared with C3 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.

Keywords: C4 photosynthesis; Dichanthelium clandestinum; Megathyrsus maximus; PEP-CK; RNA-Seq; transcriptomics..

Fig. 1.

Physiological characterization of Megathyrsus maximus and Dicanthelium clandestinum. Activity of the decarboxylation enzymes in M maximus and D. clandestinum (A); ¹³C/¹²C stable isotope ratio (B); A–C _i curves at 1500 μE (C); and light curves at 400 ppm CO₂ (D). ***P<0.001. (This figure is available in colour at JXB online.)

Fig. 2.

Shared expression based on function in NAD-ME (white set) versus all C₄ species (grey set). Up- and down-regulated functions are based on expression of functions represented by enzyme classifiers (EC) (A, B) and by Pfam domain combinations (PDC) (C, D). PPDK, pyruvate phosphate dikinase; PPase, inorganic pyrosphate phosphorylase; AMK, adenosine monophosphate kinase; PEPC, phosphoenolpyruvate carboxylase; AspAT, aspartate aminotransferase; MDH, malate dehydrogenase; ME, malic enzyme; PDH, pyruvate dehydrogenase; PEP-CK, phosphoenolpyruvate carboxykinase; AlaAT, alanine aminotransferase; CBBC, Calvin–Benson–Bassham cycle; PR, photorespiration; Asp, aspartate; PPT, phosphoenolpyruvate phosphate translocator; PS, photosynthesis; BASS2, pyruvate transporter; NHD sodium proton antiporter; all functions are listed in Supplementary Table S2 at JXB online.

Fig. 3.

Extended model for NAD-ME with high PEP-CK activity. Transport modules, consisting of one or more transporters, are shown together with the net transport through the module. Abbreviations: (1) Phosphoenolpyruvate carboxylase; (2) malate dehydrogenase; (3) NAD-dependent malic enzyme (NAD-ME); (4) pyruvate dehydrogenase kinase; (5) alanine aminotransferase; (6) pyruvate, phosphate dikinase; (7) aspartate aminotransferase; (8) aspartate oxidase and aspartate kinase; (9) phosphoenolpyruvate carboxykinase (PEP-CK); 3-PGA, 3-phosphoglyceric acid; TP, triose-phosphate; CBB, Calvin–Benson–Bassham cycle; OAA, oxaloacetic acid; RE, reducing equivalent; BASS2, pyruvate transporter; NHD, sodium proton antiporter; PPT, phosphoenolpyruvate phosphate translocator; TPT, triose-phosphate phosphate translocator; ETC, electron transfer chain. Dashed arrows represent leakage to general metabolism. (This figure is available in colour at JXB online.)