C4 photosynthesis outperforms the ancestral C3 state in a wide range of natural and agro-ecosystems by affording higher water-use and nitrogen-use efficiencies. It therefore represents a prime target for engineering novel, high-yielding crops by introducing the trait into C3 backgrounds. However, the genetic architecture of C4 photosynthesis remains largely unknown. To define the divergence in gene expression modules between C3 and C4 photosynthesis during leaf ontogeny, we generated comprehensive transcriptome atlases of two Cleomaceae species, Gynandropsis gynandra (C4) and Tarenaya hassleriana (C3), by RNA sequencing. Overall, the gene expression profiles appear remarkably similar between the C3 and C4 species. We found that known C4 genes were recruited to photosynthesis from different expression domains in C3, including typical housekeeping gene expression patterns in various tissues as well as individual heterotrophic tissues. Furthermore, we identified a structure-related module recruited from the C3 root. Comparison of gene expression patterns with anatomy during leaf ontogeny provided insight into genetic features of Kranz anatomy. Altered expression of developmental factors and cell cycle genes is associated with a higher degree of endoreduplication in enlarged C4 bundle sheath cells. A delay in mesophyll differentiation apparent both in the leaf anatomy and the transcriptome allows for extended vein formation in the C4 leaf.

SummaryAn integrative analysis of all three C4 subtypes is presented. It is shown that PEP-carboxykinase represents an addition to the NAD- or NADPH-malic enzyme subtypes but not a distinct subtype.

a b s t r a c t C 4 photosynthesis is a trait that has evolved in 66 independent plant lineages and increases the efficiency of carbon fixation. The shift from C 3 to C 4 photosynthesis requires substantial changes to genes and gene functions effecting phenotypic, physiological and enzymatic changes. We investigate the role of ancient whole genome duplications (WGD) as a source of new genes in the development of this trait and compare expression between paralog copies. We compare Gynandropsis gynandra, the closest relative of Arabidopsis that uses C 4 photosynthesis, with its C 3 relative Tarenaya hassleriana that underwent a WGD named Th-␣. We establish through comparison of paralog synonymous substitution rate that both species share this paleohexaploidy. Homologous clusters of photosynthetic gene families show that gene copy numbers are similar to what would be expected given their duplication history and that no significant difference between the C 3 and C 4 species exists in terms of gene copy number. This is further confirmed by syntenic analysis of T. hassleriana, Arabidopsis thaliana and Aethionema arabicum, where syntenic region copy number ratios lie close to what could be theoretically expected. Expression levels of C 4 photosynthesis orthologs show that regulation of transcript abundance in T. hassleriana is much less strictly controlled than in G. gynandra, where orthologs have extremely similar expression patterns in different organs, seedlings and seeds. We conclude that the Th-␣ and older paleopolyploidy events have had a significant influence on the specific genetic makeup of Cleomaceae versus Brassicaceae. Because the copy number of various essential genes involved in C 4 photosynthesis is not significantly influenced by polyploidy combined with the fact that transcript abundance in G. gynandra is more strictly controlled, we also conclude that recruitment of existing genes through regulatory changes is more likely to have played a role in the shift to C 4 than the neofunctionalization of duplicated genes. DATA: The data deposited at NCBI represents raw RNA reads for each data series mentioned: 5 leaf stages, root, stem, stamen, petal, carpel, sepal, 3 seedling stages and 3 seed stages of Tarenaya hassleriana and Gynandropsis gynandra. The assembled reads were used for all analyses of this paper where RNA was used. http://www.ncbi.nlm.nih.gov/Traces/sra/?study=SRP036637, http://www.ncbi.nlm.nih.gov/ Traces/sra/?study=SRP036837 den Bergh), canan.kuelahoglu@uni-duesseldorf.de (C. Külahoglu), andrea.braeutigam@uni-duesseldorf.de (A. Bräutigam), jmh65@cam.ac.uk (J.M. Hibberd), andreas.weber@uni-duesseldorf.de (A.P.M. Weber), zhuxinguang@picb.ac.cn (X.-G. Zhu), eric.schranz@wur.nl (M. Eric Schranz).