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

Andrea Bräutigam,Canan Külahoglu,Alisandra K Denton,Martin J Lercher,Andreas P M Weber,Janina Maß

doi:10.1093/jxb/erw463

Abstract

The high efficiency of C4 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 C4 tissues along a developing leaf gradient. This method treats both C4 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 C4 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 C4 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.

Highlights

Specialization and coordination between two cell types improves photosynthetic efficiency in most C4 photosynthetic plants
In the second, targeted at capturing unadulterated metabolite levels, two 8 cm slices were harvested in full illumination and quenched in liquid nitrogen within a second of cutting
M and bundle sheath (BS) tissues were enriched using a method modified from Stitt and Heldt (1985) that capitalizes on the distinct physical properties of M and BS cells to enrich them in different separation fractions as ground tissue is filtered through serially smaller meshes over liquid nitrogen

Summary

Introduction

Specialization and coordination between two cell types improves photosynthetic efficiency in most C4 photosynthetic plants. Most C4 plants shuttle carbon from a surrounding mesophyll (M) tissue into a surrounded bundle sheath (BS) tissue (Hatch, 1987). The shuttling concentrates CO2 around the carbon fixing enzyme, Rubisco, thereby suppressing photorespiration and increasing photosynthetic efficiency. 148 | Denton et al. C4 plants in photorespiration-inducing (e.g. hot and arid) environments (Schulze et al, 1996). The high photosynthetic efficiency and stress tolerance of C4 species has led to interest in engineering the trait. The complexity of the trait—with many changes to anatomy and metabolism beyond the core biochemical pump—makes this an ambitious goal, which will require a full systems-level understanding of both the mature C4 trait and its development to be achieved (Sage and Zhu, 2011)

Methods

Results

Conclusion