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Investigating the NAD-ME biochemical pathway within C4 grasses using transcript and amino acid variation in C4 photosynthetic genes

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Abstract

Expanding knowledge of the C4 photosynthetic pathway can provide key information to aid biological improvements to crop photosynthesis and yield. While the C4 NADP-ME pathway is well characterised, there is increasing agricultural and bioengineering interest in the comparably understudied NAD-ME and PEPCK pathways. Within this study, a systematic identification of key differences across species has allowed us to investigate the evolution of C4-recruited genes in one C3 and eleven C4 grasses (Poaceae) spanning two independent origins of C4 photosynthesis. We present evidence for C4-specific paralogs of NAD-malic enzyme 2, MPC1 and MPC2 (mitochondrial pyruvate carriers) via increased transcript abundance and associated rates of evolution, implicating them as genes recruited to perform C4 photosynthesis within NAD-ME and PEPCK subtypes. We then investigate the localisation of AspAT across subtypes, using novel and published evidence to place AspAT3 in both the cytosol and peroxisome. Finally, these findings are integrated with transcript abundance of previously identified C4 genes to provide an updated model for C4 grass NAD-ME and PEPCK photosynthesis. This updated model allows us to develop on the current understanding of NAD-ME and PEPCK photosynthesis in grasses, bolstering our efforts to understand the evolutionary ‘path to C4’ and improve C4 photosynthesis.

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Acknowledgements

This research was funded by the Australian Government through the Australian Research Council Centre of Excellence for Translational photosynthesis under the following Grant: CE140100015.

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Authors and Affiliations

  1. Hawkesbury Institute for the Environment, University of Western Sydney, Locked Bag 1797, Penrith, NSW, 2751, Australia

    Alexander Watson-Lazowski, Alexie Papanicolaou & Oula Ghannoum

  2. Research School of Biology, Australian National University, Canberra, ACT, 2601, Australia

    Robert Sharwood

  3. ARC Centre of Excellence for Translational Photosynthesis, Australian National University, Canberra, ACT, 2601, Australia

    Alexander Watson-Lazowski, Alexie Papanicolaou, Robert Sharwood & Oula Ghannoum

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  1. Alexander Watson-Lazowski
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Correspondence to Alexander Watson-Lazowski.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11120_2018_569_MOESM1_ESM.xlsx

Supplementary material 1. Supplementary 1—Transcript abundance of all genes investigated from additional replicates. Normalised transcript abundances (transcripts per million) for each gene investigated within this manuscript from additional replicates to confirm gene expression patterns. (XLSX 26 KB)

11120_2018_569_MOESM2_ESM.fasta

Supplementary material 2. Supplementary 2—Sequence alignments. All alignments used throughout manuscript as nucleotides. Nucleotide sequences were aligned via MAFFT translational alignments, using a 1.53 gap penalty and 0.123 offset value, with a G-INS-i algorithm and BLOSUM62 scoring matrix. (FASTA 193 KB)

11120_2018_569_MOESM3_ESM.docx

Supplementary material 3. Supplementary 3—Command used to create ultrametric tree. The commands run using the custom command block of MrBayes, within Geneious. (DOCX 13 KB)

11120_2018_569_MOESM4_ESM.docx

Supplementary material 4. Supplementary 4—List of gene ID’s. A list of the sequences referenced within this manuscript which come from species with published genomes, and which published ID’s they correspond to. (DOCX 16 KB)

11120_2018_569_MOESM5_ESM.jpg

Supplementary material 5. Supplementary 5—AspAT phylogenetic tree. Protein phylogenetic tree of all AspAT transcripts identified as well as known A. thaliana and Z. mays AspAT genes. Tree was built using protein alignments and RaxML (CAT BLOSUM62 model) with a bootstrap of 1000. Branch labels correspond to consensus branch support (%) and scale represents substitution rate. Tip labels correspond to the species used; PB - P. bisulcatum, PC – P. coloratum, PV – P. virgatum, PM – P. monticola, EF - E. frumentaceae, PA – P. antidotale, AX - A. fissifolius, SB – S. bicolor, CG – C. gayana, AP – A. pectinate, LF – L. fusca and LD – L. dubia. (JPG 2389 KB)

11120_2018_569_MOESM6_ESM.xlsx

Supplementary material 6. Supplementary 6—Transcript abundance of SLC25 transporters and C4 genes. Normalised transcript abundances (transcripts per million) for each gene investigated within the species investigated. (XLSX 26 KB)

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Watson-Lazowski, A., Papanicolaou, A., Sharwood, R. et al. Investigating the NAD-ME biochemical pathway within C4 grasses using transcript and amino acid variation in C4 photosynthetic genes. Photosynth Res 138, 233–248 (2018). https://doi.org/10.1007/s11120-018-0569-x

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