Does C4 photosynthesis occur in wheat seeds?

In our humble opinion, the debate about the conclusions drawn in a recent paper regarding the possibility of C4 photosynthesis being active in wheat seeds is one of the more interesting scientific debates raging at present.

The debate started with a paper published by Rangan et al last year which described findings they had made about differential expression of genes implicated in C4 photosynthesis within wheat seeds. These expression levels differed to the gene expression levels in other parts of the plant. We wrote an article describing the research here.

The response to that article (and our post) was that the evidence provided, although novel and interesting, wasn’t enough to justify the conclusion that C4 photosynthesis was in fact occurring within the wheat seed. Noting the criticisms, we wrote an updated piece covering two published responses to the Rangan article.

But the debate hasn’t stopped there…

Back and forth we go

The journal Plant Physiology published a series of letters between the researchers who published the initial research and two researchers with the differing opinion. The series of letters outlines the main points of difference between the propositions.

In defence of the C4 pathway conclusion

The series starts with a defence of the conclusions drawn from the research. Citing the earlier work of Bort et al (1995), a paper which described labeled carbon assimilation differences between leaves and seeds of wheat and barley and which found no significant difference between the location of the assimilated labeled carbon, the defenders argued that the research did not distinguish between carbon assimilated in the glumes covering the seed (the Bort et al paper) and carbon assimilated in the pericarp (the Rangan et al research).


Dissection of Wheat Glume showing glume and grain.


Figure from article showing cross-section of wheat seed, particularly the pericarp.

The distinction between the two asserted is that the Rangan et al research shows that pericarp, as opposed to the glume, demonstrates elevated C4 gene transcription.

Further, it is suggested that the carbon source supplying the pericarp is not from the capture of external CO2 but instead comes from the endosperm capturing respired carbon, the carbon being derived from bicarbonate in the developing seed tissue, and moving outwards to the pericarp for use in the asserted C4 pathway. For this reason it is suggested that the conclusions drawn by Bort et al, that there was no evidence for C4 photosynthesis in the ears of C3 cereals from their labeled-carbon pulse experiment, is a false basis to deny their conclusion as it failed to account for this inside-out carbon delivery.

To the contrary

The first point raised in contradiction to the Rangan et al research repeats the earlier criticism – that although the gene expression profiles reported in the disputed paper are novel, interesting and worthy of further research, they alone are not enough to justify the conclusions made. To be able to make such a conclusion, evidence demonstrating flux of the metabolites through the C4 pathway. Increased expression of the relevant genes is not enough to conclude that the pathway is operating in the seed particularly as all the genes involved in C4 photosynthesis are also expressed in C3 plants.

Although the evidence for the existence of the pathway in wheat seeds is scarce and contradictory, the defenders of the rebuttal suggest that the Bort et al article is evidence that the PEP carboxylase activity assists in intermediate reactions in metabolic pathways other than C4 photosynthesis.

Further, the evidence in the Rangan et al paper suggested that the expression of genes encoding the Rubsico enzyme was minimal. The contention is that, if the increased transcription of C4 genes is relied upon as evidence of increased activity within the pathway, reduced transcription of this vital photosynthetic enzyme must lead to the conclusion that the increase in PEP carboylase activity must be in aid of some pathway other than a photosynthesis pathway. Coupled with this, the low concentration of CO2 in the pericarp, where the Rubisco is located, is contrary to the high concentration of CO2 around Rubsico common in C4 tissues. Accordingly, expending energy to run a C4 pathway when the carbon would flow to Rubsico within the pericarp without the need for the pathway is contrary to expected energy conservation measures.

…and back again

In response to the criticism that evidence of flux through the pathway is lacking, the defenders of the Rangan et al paper suggest that such evidence has already been reported in a 1976 paper in the C3 intermediate barley and the relevant proteins isolated in research reported in 1986. It is argued that their research has showed that C4 specific versions of the genes were expressed in the seeds compared to the C3 versions expressed in the leaves.

In relation to the Rubisco levels, the Rubsico levels reported in the paper were for the whole seed, not just the pericarp and earlier work has shown that Rubsico is specifically expressed in the pericarp with very limited levels in the endosperm. As such, it is argued that the although the levels were rather limited in the seed as a whole, they are in fact present in high concentrations in the pericarp, the site where the C4 pathway is said to concentration CO2 levels.

Returning to the problem of the Bort et al paper, the researchers again suggest that the experiment conducted in that research could not have tested for the possibility of a C4 pathway supplying CO2 to the pericarp from within the seed, after which it would be utilised by Rubsico concentrated in the pericarp. Accordingly, they argue that the conclusions of that study fail to invalidate their conclusions.

As for the possibility that CO2 could more efficiently diffuse to the pericarp, it is contended that if that was the case it would not be concentrated enough at the site of the Rubisco and would therefore all too easily escape the seed. Their contention is that the presence of a C4 pathway in the seed is necessary for adequate CO2 concentration to the pericarp-located Rubisco.

…and one last response

The final of the letters concedes that the labeling of CO2 inside the endosperm may yield different results compared to the labeling study in the Bort et al paper, but cites the 1976 research as using isolated pericarps in their experiment and therefore doesn’t provide appropriate evidence support the C4 pathway inside the endosperm.

In relation to the diffusion versus C4 pathway dispute, it is suggested that any CO2 which is not passed from the endosperm to the pericarp through intermediates in the pathway would diffuse outwards following Fick’s law whether or not it moves through the tissue as malate. If it moved as malate, again the cost of such a pathway would be more than if it moved by simple diffusion.


The last paragraph of the final letter sums up the research as it stands and the debate about the possible conclusions that can be drawn. The research in the Rangan paper provides “an exiting new piece of the puzzle” in the quest to understand how wheat seeds increase carbon gain. It would seem that to put the debate to rest, the flux of the carbon through a C4 pathway must be demonstrated unequivocally along with measurement the location and activity of the required enzymes.

A passionate and reasoned debate over plant science which is wonderful to see.


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