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The high efficiency of C 4 photosynthesis relies on spatial division of labor, classically with initial carbon fixation in the mesophyll and carbon reduction in the bundle sheath. By employing grinding and serial filtration over liquid nitrogen, we enriched C 4 tissues along a developing leaf gradient. This method treats both C 4 tissues in an integrity-preserving and consistent manner, while allowing complementary measurements of metabolite abundance and enzyme activity, thus providing a comprehensive data set. Meta-analysis of this and the previous studies highlights the strengths and weaknesses of different C 4 tissue separation techniques. While the method reported here achieves the least enrichment, it is the only one that shows neither strong 3' (degradation) bias, nor different severity of 3' bias between samples. The meta-analysis highlighted previously unappreciated observations, such as an accumulation of evidence that aspartate aminotransferase is more mesophyll specific than expected from the current NADP-ME C 4 cycle model, and a shift in enrichment of protein synthesis genes from bundle sheath to mesophyll during development. The full comparative dataset is available for download, and a web visualization tool (available at http://www.plant-biochemistry.hhu.de/resources.html) facilitates comparison of the the Z. mays bundle sheath and mesophyll studies, their consistencies and their conflicts., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

C(4) photosynthesis outperforms the ancestral C(3) 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 C(3) backgrounds. However, the genetic architecture of C(4) photosynthesis remains largely unknown. To define the divergence in gene expression modules between C(3) and C(4) photosynthesis during leaf ontogeny, we generated comprehensive transcriptome atlases of two Cleomaceae species, Gynandropsis gynandra (C(4)) and Tarenaya hassleriana (C(3)), by RNA sequencing. Overall, the gene expression profiles appear remarkably similar between the C(3) and C(4) species. We found that known C(4) genes were recruited to photosynthesis from different expression domains in C(3), 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 C(3) 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 C(4) bundle sheath cells. A delay in mesophyll differentiation apparent both in the leaf anatomy and the transcriptome allows for extended vein formation in the C(4) leaf., (© 2014 American Society of Plant Biologists. All rights reserved.)

C₄ photosynthesis represents the most productive modes of photosynthesis in land plants and some of the most productive crops on the planet, such as maize and sugarcane, and many ecologically important native plants use this type of photosynthesis. Despite its ecological and economic importance, the genetic basis of C₄ photosynthesis remains largely unknown. Even many fundamental aspects of C₄ biochemistry, such as the molecular identity of solute transporters, and many aspects of C₄ plant leaf development, such as the Kranz anatomy, are currently not understood. Here, we review recent progress in gaining a mechanistic understanding of the complex C₄ trait through comparative evolutionary analyses of C₃ and C₄ species., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We systematically analyzed a developmental gradient of the third maize (Zea mays) leaf from the point of emergence into the light to the tip in 10 continuous leaf slices to study organ development and physiological and biochemical functions. Transcriptome analysis, oxygen sensitivity of photosynthesis, and photosynthetic rate measurements showed that the maize leaf undergoes a sink-to-source transition without an intermediate phase of C(3) photosynthesis or operation of a photorespiratory carbon pump. Metabolome and transcriptome analysis, chlorophyll and protein measurements, as well as dry weight determination, showed continuous gradients for all analyzed items. The absence of binary on-off switches and regulons pointed to a morphogradient along the leaf as the determining factor of developmental stage. Analysis of transcription factors for differential expression along the leaf gradient defined a list of putative regulators orchestrating the sink-to-source transition and establishment of C(4) photosynthesis. Finally, transcriptome and metabolome analysis, as well as enzyme activity measurements, and absolute quantification of selected metabolites revised the current model of maize C(4) photosynthesis. All data sets are included within the publication to serve as a resource for maize leaf systems biology.

C4 photosynthesis represents the most productive modes of photosynthesis in land plants and some of the most productive crops on the planet, such as maize and sugarcane, and many ecologically important native plants use this type of photosynthesis. Despite its ecological and economic importance, the genetic basis of C4 photosynthesis remains largely unknown. Even many fundamental aspects of C4 biochemistry, such as the molecular identity of solute transporters, and many aspects of C4 plant leaf development, such as the Kranz anatomy, are currently not understood. Here, we review recent progress in gaining a mechanistic understanding of the complex C4 trait through comparative evolutionary analyses of C3 and C4 species. [Copyright &y& Elsevier]