An Update to “Evidence that C4 Photosynthesis Already Exists in an Important C3 crop”

We have previously reported on an article in Nature Scientific Reports (“the Rangan paper”) that indicated the possibility that C4 photosynthesis was being carried out in the grain of wheat, an important C3 crop that belongs to a clade with no evidence that C4 photosynthesis has ever evolved within it. As C4 photosynthesis is more efficient in fixing CO2 than C3 photosynthesis, the possibility of the pathway being active in such an crop would give hope that it could be manipulated to improve its growth properties and that other important food crops.

Shortly after we posted our article it was pointed out that, although the evidence gathered in that paper are a useful analysis of gene transcript data, the presence of C4 gene transcripts should not amount to the conclusion that there is an active C4 biochemical pathway in the wheat grain. In the last week, two articles have appeared rebutting the conclusions of the paper.

“Seeds of C4 photosynthesis”

This article by Julian Hibberd and Robert Furbank in Nature Plants points out that although what is presented in the Rangan paper provides an interesting in-depth assessment of the transcripts derived from the wheat grain, the transcript data by itself doesn’t evidence a functioning pathway.

Whether the transcribed proteins are active and to what extent they are active cannot be evidenced solely from transcript data. If they are active, whether they are linked appropriately to form the C4 pathway for metabolite flux also needs to be evidenced if we are to accept the possibility of a functioning pathway.

The Rangan paper also cited differences in particular cell types within the pericarp of wheat grain and combine these differences with the abundance of C4 gene transcripts to hypothesize the possible existence of the two-celled C4 system. There were two problems with this hypothesis:

  1. No cell specific transcript evidence is given to support the assertion, let alone the protein and metabolite data. Although there are a number of different types of cells in the pericarp, the transcript analysis does not distinguish between these types of cells which would help confirm or deny a difference in transcript abundance between the types of cells; and
  2. Both the Hibberd and Furbank paper and the paper discussed below cited a protein labelling experiment which showed the PEPC protein (an important protein in the C4 pathway) is not found in the pericarp but rather the aleurone layer.

The paper concludes by saying that the transcript data isn’t enough to form a conclusion that engineering C4 photosynthesis in wheat will be any easier in wheat than in any other crop.

“Poor Evidence for C4 Photosynthesis in the Wheat Grain”

This article in Plant Physiology make the same criticisms about the lack of biochemical evidence supporting the claim of C4 photosynthesis in the wheat grain, the lack of evidence showing which cells within the grain are the origin of the transcripts obtained and that previous immunolabeling studies have shown PEPC being localised in the aleurone layer and endosperm, not the pericarp as suggested by Rangan et al.

A further criticism of the Rangan paper was the assertion made that the increase in PEPC and decrease in RuBisco transcripts is evidence of C4 photosynthesis. Again, the lack of biochemical evidence supporting an active pathway was noted. However, the authors also note that C4 photosynthesis still requires the use of RuBisco to assimilate the CO2 concentrated by PEPC and that an approximate ratio of one-to-one is required. Therefore, an over-abundance of PEPC compared to RuBisco doesn’t result in the increased photosynthetic efficiency we relate with C4 photosynthesis.

And a bit more…

We contacted Professor Robert Furbank, co-author of the Nature Plants letter, who was kind enough give us some insight into the issue, its historical context and how big a discovery it would be if C4 photosynthesis existed in wheat grain:

“It has been known since Tom ap Rees’ work in the 1970’s that a suite of C4 enzymes are present in C3 seeds but the flux is into amino acid synthesis and gluconeogenesis.

What I find interesting in the context of the Rangan paper is that the same mistakes were made which delayed the discovery of the C4 pathway by a decade. Careful measurements of photosynthetic flux which can separate a respiratory role of PEPC and other C4 enzymes from a photosynthetic one are required.  To ensure that RNAseq does not become the emperor’s new clothes, we must combine biochemistry, physiology and our new next gen sequencing tools to build a cogent and robust story.

I would be very surprised in C4 photosynthesis existed in grains.  The evolution of C4 was to concentrate CO2 around rubisco in a low CO2 atmosphere, which is certainly not the case in a developing cereal grain.  Even in the glumes, there is hard evidence that the initial evidence for C4 photosynthesis was flawed.”

Conclusion

The Hibberd and Furbank response to the Rangan paper acknowledges that the transcript analysis performed and reported on provide interesting evidence regarding differences in gene expression between different plant tissue, but warn that the gap between gene transcription and functioning protein pathways is something that must be bridged before the type of conclusion made by Rangan et al. can be made with confidence.

We must also admit that, in our post on the Rangan paper, our enthusiasm for the conclusions presented was greater than our critical analysis of evidence supporting it. We thank Steven Burgess and Julian Hibberd for their tweets pointing out issue with the conclusions drawn and Robert Furbank for taking the time to talk to us. In future, we will improve on our analysis of new articles and will seek out comments from relevant experts when significant claims are being made.

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3 thoughts on “An Update to “Evidence that C4 Photosynthesis Already Exists in an Important C3 crop”

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