Evidence that C4 Photosynthesis Already Exists in an Important C3 Crop

“The presence of all C4 specific genes in the genome confirms that natural selection may have already explored the options being considered by plant breeders.”

Rangan, P, Gurtado, A, & Henry, RJ, 2016. “New Evidence for Grain Specific C4 Photosynthesis in wheat”, Scientific Reports, vol 6. 31721; doi:10.1038/srep31721 (2016)

Edit: After publishing this article it has been helpfully pointed out in a series of tweets that the evidence of C4 photosynthesis in wheat obtained by these researchers doesn’t confirm that C4 does exist in the grain. Evidence of a functional metabolic C4 pathway is still required if the conclusion of the paper is to be accepted. Thank you to the people you helped to clarify. 

The above quote is buried under the sub-heading “varied expression pattern between wheat genomes” in this significant article published Nature’s Scientific Reports.

We have written a number of articles about C4 photosynthesis at Legume Laboratory, including;

Therefore, we wont rehash how significant an effect the ability to convert food crops from using C3 photosynthesis to the more efficient C4 photosynthesis and what we already know about it, save that the authors of this article suggest that a 30% increase in wheat yield is possible if the crop was converted to C4 photosynthesis due to the resultant improvements in water and nitrogen use efficiency.

But what these three researchers found could greatly assist the efforts of engineering or breeding crops with this important trait.

Taking 35 genotypes of wheat, they performed a trancriptome analysis on the developing grains at 14 days and 30 days after the anthesis (first opening of a flower bad, marking the start of the flowering period) and leaves to look at the variation in gene expression at the two points in time and in the different tissue. The transcripts, after being converted to cDNA and sequenced, were mapped back to the genome to ascertain the transcribing genes in the developing grains, specifically looking for genes related to C4 photosynthesis.

What was found

The authors describe three C4 photosynthesis subtypes based on particular enzymatic pathways, being the:

  1. NADP-dependent malic enzyme (“NADP-ME”);
  2. NAD-ME; and
  3. Phsophoenolpyruvate carboxylase (“PEPCK”).

These pathways, which differ from the RuBisCO common in C3 photosynthetic cells, are usually found in the Kranz Anatomy arrangement of cells found in most C4 plants, although single celled C4 photosynthesis has been found.

The transcriptome analysis performed focused on the searching for the presence of genes encoding the enzymes involved in these particular pathways given that this would be a good indicator of a different form of photosynthesis than the C3 photosynthesis taking place in the leaves of wheat crops.

Molecular evidence

What was found was the presence of all the genes (including typical isotypes) required for NAD-ME C4 photosynthesis in the caryopsis of the wheat grains, identifying their location in the genome and differences in expression rates at the different stages of development of the caryopsis and differences in expression in the caryopsis and the leaves.

  • Phosphoenolpyruvate carboxylase – was shown to be transcribed 125 times more compared to that in leaves of the crop. Further, working on the knowledge that RuBisCO transcripts were significantly reduced in C4 cells, they quantified RuBisCO transcription and found a 76 fold reduction in its expression in the caryopsis.
  • Aspartate aminotransferase – there were six copies of this gene in the cDNA libraries  but only two were the C4 types, both of which had an increase in transcription at 14 days post anthesis.
  • Malate dehydrogenase – two genes were found, with one of the two copies, the one thought to be involved in C4 photosynthesis, being differentially expressed in the caryopsis compared to leaves.
  • Malic enzyme coding gene – two copies were found, one of which (the mitochondrial targeted copy which supports C4 photosynthesis) was up-regulated in the caryopsis whilst the other was up-regulated in leaves.
  • Alanine transaminase – the gene involved in converting pyruvate to alanine in bundle sheath cells (and converting alanine to pyruvate in mesophyll cells) in NAD-ME photosynthesis reactions, was found in two copies. One of these copies was found to be expressed at a higher rate in the developing caryopsis.
  • Pyruvate, orthophosphate dikinase – comparing the expression of the gene between leaf and grains showed greater expression in the grain.

Of these six genes, phosphoenolpyruvate carboxylase (ppc) and alanine transaminase (gpt) require information about their sequence to determine whether the gene is involved in the C3 or C4 pathway (our article “Can We Synthetically Engineer C4 Photosynthesis” mentioned that many genes involved in C4 photosynthesis already exists in C3 crops but was used for other reactions).

The transcription analysis showed that gpt was expressed in similar amounts in both forms in all tissues of the crop and wasn’t analysed any further.

However, in relation to ppc, it was known that a substitution of an Arginine amino acid at position 884 in the C3 enzyme was well conserved, while C4 transcripts have been shown to contain Glycine at this position. Analysis of their transcripts showed neither Arginine or Glycine at this position, prompting further research into a number of related C4 crops and their alterations at this amino acid. What they found was that while C3 transcripts were conserved with Arginine at position 884, C4 crops related to wheat had been found with Serine, Glutamine, Glycine or Isoleuvine at the position. Therefore, the researchers suggested that transcripts with the conserved amino acid at this position were likely C3 genes while variability at this position, which may explain the improved efficiency of C4 photosynthesis, indicated a non-C3 photosynthetic use of the enzyme.

Cytological evidence

Not content with relying on the molecular evidence supporting a theory of C4 photosynthesis in the wheat grains, the researchers looked at previous research of physiological differences in cell composition of the grains. They drew on previous research that showed differences in the cross and tube cells the comprise the pericarp, particularly the differences in the number of chloroplast grana stacked in cross cells compared to tube cells. This division of labour in the photosynthesis process between the two cell types, similar to the division between bundle sheath and mesophyll cells in C4 plants, combined with the transcription of C4 specific genes targeted to this layer of the grain, led to the assertion of the researchers of the existence of C4 photosynthesis specifically in the grain compared to the C3 photosynthesis in the plant leaves.


Figure 4 from Article showing differences in cross cells and tube cells in pericarp of wheat plants.

Evolutionary evidence

The sign of good research – triple checking the assertion that has been formulated. Not content with the molecular and cytological evidence, the researchers assessed the evolution of wheat within its genus to look at the evolutionary plausibility of the existence of C4 photosynthesis in the species.

This analysis contributed to the finding of the differing amino acids at position 884 of the ppc gene transcript described above. The lack of conservation at this amino acid position was extracted from the finding that wheat and related species held 5 copies of the ppc gene, one of which had a differing amino acid at this position when compared to the other 4 with the C3-conserved Arginine residue.

Evolutionarily, the researchers traced back the altered amino acid through the genus and suggested that the evolution of C4 photosynthesis has occurred on four separate occasions. Relating their finding to the types of photosynthesis found in the tribe of which wheat belongs (Triticeae), other members of the tribe contain the same number of ppc genes, all of which have one C4 copy of the gene, whilst the evolution of Brachypodium, which branched off from this line before the evolution of the species with C4 photosynthesis, remains a C3 plant with all five copies of the gene containing the conserved amino acid (see table 2 of the article).

How does this help us?

The conclusion suggests that the simple assignment of a category of photosynthesis to which a particular plant belongs to may not be correct or helpful to further research. As highlighted in this paper, a crop which has forever been thought of as a C3 crop due to an analysis of its leaves may miss the C4 photosynthesis busily converting light in another part of the plant.

What is also means is that the possibility of engineering C4 photosynthesis into C3 crops may be less a matter of forcing new circuitry into leaves and may instead be more targeted at finessing the already present C4 genes into being transcribed throughout the plant.

A very exciting find by these three researchers.


4 thoughts on “Evidence that C4 Photosynthesis Already Exists in an Important C3 Crop

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