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

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.

Comparative transcriptome atlases reveal altered gene expression modules between two Cleomaceae C3 and C4 plant species.

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.

Azolla domestication towards a biobased economy?

Due to its phenomenal growth requiring neither nitrogen fertilizer nor arable land and its biomass composition, the mosquito fern Azolla is a candidate crop to yield food, fuels and chemicals sustainably. To advance Azolla domestication, we research its dissemination, storage and transcriptome. Methods for dissemination, cross-fertilization and cryopreservation of the symbiosis Azolla filiculoides-Nostoc azollae are tested based on the fern spores. To study molecular processes in Azolla including spore induction, a database of 37 649 unigenes from RNAseq of microsporocarps, megasporocarps and sporophytes was assembled, then validated. Spores obtained year-round germinated in vitro within 26 d. In vitro fertilization rates reached 25%. Cryopreservation permitted storage for at least 7 months. The unigene database entirely covered central metabolism and to a large degree covered cellular processes and regulatory networks. Analysis of genes engaged in transition to sexual reproduction revealed a FLOWERING LOCUS T-like protein in ferns with special features induced in sporulating Azolla fronds. Although domestication of a fern-cyanobacteria symbiosis may seem a daunting task, we conclude that the time is ripe and that results generated will serve to more widely access biochemicals in fern biomass for a biobased economy.

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

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.

Quantitative transcriptome analysis using RNA-seq.

RNA-seq has emerged as the technology of choice to quantify gene expression. This technology is a convenient accurate tool to quantify diurnal changes in gene expression, gene discovery, differential use of promoters, and splice variants for all genes expressed in a single tissue. Thus, RNA-seq experiments provide sequence information and absolute expression values about transcripts in addition to relative quantification available with microarrays or qRT-PCR. The depth of information by sequencing requires careful assessment of RNA intactness and DNA contamination. Although the RNA-seq is comparatively recent, a standard analysis framework has emerged with the packages of Bowtie2, TopHat, and Cufflinks. With rising popularity of RNA-seq tools have become manageable for researchers without much bioinformatical knowledge or programming skills. Here, we present a workflow for a RNA-seq experiment from experimental planning to biological data extraction.