Towards an integrative model of C4 photosynthetic subtypes: insights from comparative transcriptome analysis of NAD-ME, NADP-ME, and PEP-CK C-4 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

Azolla domestication towards a biobased economy?

Brouwer P, Bräutigam A, Külahoglu C, et al. Azolla domestication towards a biobased economy? New Phytologist. 2014;202(3):1069-1082.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 37649 unigenes from RNAseq of microsporocarps, megasporocarps and sporophytes was assembled, then validated. Spores obtained year-round germinated in vitro within 26d. In vitro fertilization rates reached 25%. Cryopreservation permitted storage for at least 7months. 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

Comparative Transcriptome Atlases Reveal Altered Gene Expression Modules between Two Cleomaceae C-3 and C-4 Plant Species

Külahoglu C, Denton AK, Sommer M, et al. Comparative Transcriptome Atlases Reveal Altered Gene Expression Modules between Two Cleomaceae C-3 and C-4 Plant Species. Plant Cell. 2014;26(8):3243-3260.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

Fig S1-7 All Supplemental Figures

Figure S1. Setup of developmental slices, enzyme/metabolite data deconvolution method, and validation of separation method. Figure S2. Metabolite levels in BS and M of the developmental slices (full data in dataset S1). Figure S3. Contextual data of developmental section pairing, and library complexity for inter-study comparison. Figure S4. Transcript coverage of genes (or their orthologs) that were significantly enriched in the BS in Chang_2012, but in M in here Denton_2016. Figure S5. Transcript coverage of examples genes from conflict set 1 (definition and gene list in dataset S5). Figure S6. Expression of marker genes/categories used to evaluate the co-purification of additional tissues. Figure S7. AlaAT and 40S ribosome distributions

Seaton Computing Center Before Renovation_03

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S1, Enzyme activity and metabolite abundance

Enzyme activity and metabolite abundances from enriched bundle sheath, and mesophyll tissues along a developming Zea mays leaf. Data provided from two havests. Fast=two, 8cm slices, less than 1s to liquid nitrogen. Gradient=five, 4cm slice, ~10s to liquid nitrogen. Data is provided both in raw, and fully normalized ("deconvoluted", see methods of paper) form

Supplemental_Table2 Numbers of Signifiant Differences

Quantification of the number of significant differences in expression found between BS and M in each study and developmental category. Significance was calculated with edgeR for all studies, and additionally with contamDE for Denton_2016

S2, Functional enrichments.

Significant functional enrichment in gene sets that were otherwise informative in understanding inter-study differences. First four data columns represent a contingency table with "S" standing for "set (of genes)" and "C" standing for "Category (functional)". A Fisher's exact test was used to determine significance, and the false discovery rate calculated using the Benjamini Yekutili method. Remaining columns supply information on the functional category, details of gene set, and direction of enrichment

Supplemental Table1 Bioinformatis Parameters

Non-default parameters used for bioinformatics tools in this study