C4 Photosynthesis Evolution: why some intermediates may not assist our understanding

C4 photosynthesis, a form of photosynthesis which converts CO2 with greater efficiency than the more common C3 variant, is widely seen as a promising trait which could be used to improve crop growth. This is especially so with the looming concerns over the effects of climate change on food production.

We have previously written about C4 photosynthesis, how plant anatomy and chemistry differs between the two forms of photosynthesis and the possibility of being able to engineer C4 photosynthesis into crop plants.

Further, we have written about an article that suggested that certain parts of wheat plants may already perform C4 photosynthesis and, in a subsequent piece, we wrote about two rebuttals to that assertion.

In the aforementioned article regarding engineering C4 photosynthesis, the use of intermediate crops as a means to assist us to discover how some C3 crops have been able to evolve into C4 crops has been suggested as a means of furthering our knowledge of C4 evolution and to assist us to convert important C3 crops into more efficient light converters.

The article we are writing about this week warns researchers that the use of C3-C4 intermediate crops for such a purpose may not hold the answers to the questions we are asking.

C3-C4 Intermediates

This report recently published in the New Phytologist starts with a short description of C4 photosynthesis and its discovery. Two of these early papers on C4 photosynthesis evolution suggested that plants containing a photosynthetic system which appears to be an intermediate between C3 and C4 photosynthesis may be a proxy for the evolution of C4 photosynthesis.

However, the authors of this paper also point out that it is possible that these intermediates may, instead of depicting an plant in the throws of an evolutionary conversion, be a hybrid plant between two closely related species where one parent uses C3 photosynthesis and the other uses C4 photosynthesis.

The problem this raises is that research of C4 evolution may be misdirected if the intermediate under study is not truly undergoing this process.

To examine this possibility, the authors of the paper looked at the evidence of both natural and experimentally generated C3-C4 hybrids and described the categories of intermediates that have been discovered.

Categories of Intermediates

Intermediates of photosynthesis were categorised in a previous article by Sage et al (2014) which distinguished four categories of intermediate:

  1. Proto-Kranz: the plant contains bundle sheath cells or Kranz-like cells which have some similar traits to C4 species such as being larger and orientated in a centripetal position.
  2. C2 type 1: contains the same characteristics as proto-Kranz cells, but which also shuttles glycine produced in the mesophyll cells during photorespiration to the Kranz-like cells where its is decarboxylated and the photorespired CO2 is concentrated and partially refixed by Rubisco. This refixation of CO2 that would otherwise be lost in photorespiration in C3 and proto-Kranz photorespiratory cells improves efficiency.
  3. C2 type 2: the same as C3 type 1 save that the refixation of CO2 appears to rely largely on enzymes normally found in C4 photosynthesis, although their expression is lower than that found in C4 and C4-like species. C2 type 1 cells, by comparison, rely largely on a glycine shuttle and C4 enzymes are much less active.
  4. C4-like: a rarely found intermediate that shows increased activity of enzymes used in the C4 photosynthetic cycle and reduced reliance on C3-active enzymes.

Adapted from the Sage article is a table which contains a comprehensive list of plant species that fit within each criteria and under which category they sit. The Sage article also provides the below diagram illustrating the evolution from C3 to C4 photosynthesis.

C3 evolution

Figure from Sage et al (2014) showing the differences between C3, C4 and each intermediate category of photosynthesis. The figure in the article contains a lengthy description of the illustration (and the article is freely available).

The possibility of photosynthetic hybrids

Hybrid intermediates were experimentally attempted prior to the discovery of naturally occurring hybrids and have since been successfully created experimentally in a number of closely related species. Experimentally created hybrids have resulted in a mix of intermediate photosynthetic systems that were scattered throughout the spectrum between C3 and C4 photosynthesis. Intermediates generated would contain some components that related to C4 photosynthetic systems but would retain some C3 properties and similar properties would be passed in varying degrees to the segregating populations generated from the experimental hybrid.

In all, the previous research demonstrated the hybrids (C3 x C4, C3 x C3-C4, and C4 x C3-C4) could be classified in accordance with the criteria contained in the Sage paper to classify naturally occurring hybrids. The problem this causes, the authors suggest, is that the hybrids cannot be differentiated from true evolutionary intermediates on the basis of the Sage criteria.

The Naturally Occurring Hybrids

Naturally occurring photosynthetic hybrids have been considered likely in a number of papers where lineages have contained C3, C4 and some intermediate species. The article provides examples of a number of papers which observed or considered likely that hybridisation is the cause of the intermediate species.

The concern raised by the authors is that studies looking at phylogenetic trees of such lineages rarely look specifically for hybridisation as a cause of the photosynthetic intermediates. Therefore, the cause of the identified intermediates may be mistaken as being due to evolutionary changes rather than being due to hybridisation.


On the basis of the similarity between experimentally created photosynthetic hybrids and natural intermediates and their inability to be distinguished from each other using the Sage criteria, the authors believe that greater care should be taken when considering which intermediate is to be used to study C4 evolution. Taking the point further, actively excluding the possibility of the intermediate under study being of hybrid rather than evolutionary basis would be best practice before embarking on its use for the purpose of studying how C4 may have evolved and linking it to research of how we may create a similar evolution.

Finally, although hybrids may not be good candidates for evolutionary study, such hybrids aren’t without any worth. The hybrids experimentally created and discussed in the papers cited by these authors could be themselves be the basis of further research that could assist us to improve photosynthetic efficiency in food crops.


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