Freeze-quenched maize mesophyll and bundle sheath separation uncovers bias in previous tissue-specific RNA-Seq data.

The high efficiency of C 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 tissues along a developing leaf gradient. This method treats both C 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 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 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.