“The C4 pathway is one of the most complicated biochemical pathways likely to be placed in front of a university undergraduate, and attempts at C4 engineering have only served to reveal additional uncertainties about how the pathway operates, how it is regulated at the level of gene expression and post-translationally, and some of the physical properties of the anatomical specialization of Kranz C4 leaves.”
Robert Furbank (2016) “Walking the C4 pathway: past, present, and future”, Journal of Experimental Botany, vol. 67, no. 14, pp. 4057 – 4066
Whilst writing our post titled “C4 Photosynthesis Evolution: Why some intermediates may not assist our understanding” we came across a 2016 paper written by Professor Furbank covering the discovery of C4 photosynthesis, the research and stories behind our understanding of this important photosynthetic trait, the gaps in our knowledge that remain to be filled and where the research may lead moving forward.
Readers of this blog may recall that Professor Furbank generously gave us the time to explain the problems in a research article proposing that C4 photosynthesis already existed in wheat which greatly assisted our updated post on the issue. He has also recently been a guest on the Australian Broadcasting Corporation’s “The Science Show” talking about C4 photosynthesis and the positive effects it could have on our food security (listen here).
Discovery of an important trait
Prior to the biochemical description of C4 photosynthesis detailed by Hal Hatch and Roger Slack in 1966, the special properties of particular plants that were later understood to use C4 photosynthesis had been documented since the turn of the 20th century. Professor Furbank’s review lists the important research articles which led the way to our current understanding, such as the discovery of Kranz anatomy, plants increased water use efficiency and increased photosynthetic rates compared other plants.
The separation between the collection and concentration of CO2 in mesophyll cells before pumping the CO2 to bundle sheath cells, where it is reduced and decarboxylated, is the most important and distinguishing trait used to differentiate C4 photosynthesis from other forms of photosynthesis. The concentration of CO2 allows Rubsico to operate at greater efficiency, reducing the competition for the active site of the enzyme that normally plays out between CO2 and O2 in non-C4 species, benefiting the growth rate and resource efficiency of the plant.
Figure from article demonstrating the Kranz anatomy (ring of purple bundle sheath (BS) cells and mesophyll cells (M) within the plant tissue. The numbers on the right of the figure denote; 1. carbonic anhydrase, 2. PEP carboxylase, 3. malate dehydrogenase, 4. NADP-malic enzyme, 5. pyruvate orthophosphate dikinase.
Unpublished data, criticisms of legitimate research findings and unnoticed publications of important research in obscure or foreign-language journals hampered early progress in discovering C4 photosynthesis until the seminal work of Hatch and Slack (who we are proud to learn are Australian scientists) in 1966.
Hatch and Slack’s 1966 research paper used 14C-labelled CO2 to study the phenomenon, applying a pulse of labeled CO2 and then illuminating the subject leaf to follow the progression of the labeled CO2 as it made its way through the photosynthetic cycle. This method is commonly known as a ‘pulse-chase’ analysis. The idea for the experiment came from similar earlier experiments conducted previously by other researchers but which had provided results that lacked certainty in the identification of the labeled compound. Whilst having chatting over a beer during the Australian Biochemical Society conference held in Hobart in 1965, Hatch and Slack decided to try their hand at replicating these earlier experiments in a manner that would reduce or remove the uncertainty contained within the earlier results and shed further light on the strange data that was being produced in these earlier works.
In something reminiscent of the oversight of Gregor Mendel’s early research on genetics in pea plants, it was discovered in the late 1960’s that a Russian researcher named Yuri Karpilov had reported similar findings from his research as that obtained by Hatch and Slack in an article he published in 1960 in a little known journal.
What wasn’t grasped at the time was that this newly discovered pathway resulted in a pool of CO2 concentrated in some cells while Rubsico, an enzyme difficult to extract, was present and operating in the separate bundle sheath cells. This discovery was slowly uncovered in a series of papers in the late 1960s and early 1970s, as was the importance of the concentration of CO2 in reducing photorespiration and its link to improved plant performance.
As our methods of isolating and probing at different cells have improved, so has our knowledge of the variety of biochemical pathways that exist within the C4 pathway. However, we are still grasping at which of these pathways provides the greatest flux of metabolites. When we acquire this understanding it will focus our attention to those pathways likely to result in the greatest improvements in plant performance and harness our attempts to engineer C4 photosynthesis in important crops.
Since the significant increase in our knowledge of this important photosynthetic pathway around the time of Hatch and Slack’s 1966 paper, genetic transformation of plants and the increase in our ability to extract and subject plant data to bioinformatics techniques has continued to push research forward. The use of green foxtail millet as a C4 model grass by the biofuels community has allowed for the development of transformation protocols to help probe deeper into the genetics, transriptomics and protein products that form the basis of this system. Further, this information is crucial to our engineering attempts aimed at improving crop productivity.
Similarly, next-generation sequencing (NGS) has revolutionized research probing the evolution of C4 photosynthesis and how they may have evolved from their C3 ancestors. Probing the differences between the genetics and gene transcripts between the differentiated cells is steadily helping us understand and pinpoint which genes are absolutely necessary for a functioning C4 pathway. Analysis of C4 meristem tissue as it grows and the adjustment of gene transcription through this growth is also assisting in deciphering what gene regulation is required if we are to successfully install C4 photosynthesis into important C3 crops.
Where to now?
There is still important research to be done and discoveries to be made in C4 photosynthesis research.
Quantifying the amount of fixed CO2 that is leaked from mesophyll cells, under what conditions greater or lesser amounts of CO2 is leaked and what physical properties of the bundle sheath cells may change the rate of flux are all questions that remain to be answered if we are to successfully engineer the system into C3 crops. As with many areas of research, new tools to extract the information and avoid the uncertainty that plagues the more indirect methods of investigation we are currently limited to will greatly assist the research.
Further information about the positioning of important organelles within the cells and the ratios of bundle sheath cells to mesophyll cells are yet to be understood and which could prove to hamper engineering efforts.
Finally, despite the significant leap in the genetic and transciptomic knowledge we have seen of recent times, there is still only a developing understanding of gene regulation in the growing plant tissue to ensure the separation of the two parts of the carbon fixation and decarboxylation process within the tissue. Important transcription elements specific to this demarcation of activity between the cells are still being investigated and isolated.
Significant research effort is being funded to enable the discovery of these important missing pieces of information and into attempting to transfer C4 photosynthesis into important C3 crops. The C4 Rice Project, funded by the Bill and Melinda Gates Foundation, is one such effort that looks very promising.
So, can we supercharge a VW to perform like a Porsche? The progress being made and the dedication to the problem must lead us to a sense of optimism that it will happen within our lifetimes.