“Photosynthesis as the engine for life on earth has high engineering potential, which has not yet been fully exploited…By step-wise identification of all the components needed for engineering, it will eventually become possible to employ this powerful machinery to increase yields for the future.”
Schuler, ML, Mantegazza, O & Weber, APM, 2016, ‘Engineering C4 photosynthesis into C3 chassis in the synthetic biology age’. The Plant Journal, vol. 87, pp. 62
These lines from the conclusion of the review we write about here are indicative of why so much effort is being put into understanding the more productive C4 photosynthetic system and working to increase important crop yields with it.
Schuler, Mantegazza and Weber’s article in the special issue of The Plant Journal on plant synthetic biology provides an excellent overview of the current status, significant hurdles and possible solutions to those problems of the current research aimed at bolstering rice yield by converting it from the common C3 photosynthesis system to the more efficient C4 system. We’ve previously written about C4 photosynthesis here and here.
C4 photosynthesis
C4 photosynthesis has evolved independently at least 66 times and is likely linked to a sudden drop in atmospheric CO2 levels sometime in the past. It is characterised by the concentration of CO2 around Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), the carbon-assimilating enzyme, reducing the competition that CO2 has with O2 to interact with the enzyme. More CO2 means greater growth and reduced photorespiration, an energy requiring process that is used to remove the O2 reaction products.
The concentration of CO2 in C4 photosynthesis is usually caused by a two-celled (but one-celled is possible) distribution of the process of fixing carbon and the process of reducing it. The two-celled system combines mesophyll (M) cells, which take up the CO2 from the leaf air space, and the bundle sheath (BS) cells, where the Rubisco enzymes reside, the final destination of CO2 for fixation and entry into the Calvin-Benson cycle. These two cells are arranged in concentric layers (called ‘Kranz Anatomy’) around leaf veins, maximising the contact between the two types of cells and increasing the transport of the molecules between them.
M cells convert CO2 to bicarbonate and then into the 4 carbon compound oxaloacetate via an enzyme that doesn’t react with oxygen. The modified compound is then passed to the BS cells where it is reformed into CO2 and fixed by Rubisco to enter the Calvin-Benson cycle.
Basically, by assimilating CO2 away from Rubisco, the plant reduces the ability of Rubisco to interact with O2 and instead it is steadily fed with CO2 from the M cells.
Of course, this description of the process is simplified and although most of the process and main enzymes that carry out the process are known, there are still gaps in our knowledge.
Recent Advances
The gathering of increasing amounts of genomic, trascriptomic and metabolimic data continue to improve our knowledge of C4 photosynthesis, how it evolved and how we might transition C3 crops to use the more efficient carbon fixation method.
Important C4 crop species have had their genomes sequenced and quantitative analysis of transcriptomes have begun to unravel the mystery behind the genes upregulated and downregulated, and the stage of development that these regulatory differences occur, that lead the formation of the Kranz anatomy. What we are finding is that many of the genes involved in C4 photosynthesis exist in C3 plants but are differently regulated at early stages to differentiate the BS and M cells, enable high throughput of metabolites between the cells and to increase the size of vascular tissue to support the increased activity.
Engineering C4 photosynthesis
Our initial attempts to engineer C4 photosynthesis relied on over-expressing one or more enzymes in C3 plants. However, given the enzymes involved in the C4 system are used in the C3 system in multiple alternative pathways, the effects of over-expression were multiple, varied and didn’t have the desire result. The compartmentalisation of reactions, whether in the single or two-celled reactions that make up the distinctive photosystem, is complex.
The notion of being able to engineer C4 photosynthesis is comforted by a number of factors:
- The main enzymes are already present in C3 photosynthesis;
- Characteristics such as the passing of metabolites between cells is seen in C3 species such as tobacco plants; and
- Nature has done it herself in the past on multiple, independent occasions.
But the authors of the paper also note a number of engineering steps that need to be accomplished if we are re-enact evolution ourselves;
- Higher order veins need to be initiated in plants (it previously being shown that such physical properties were already evolved in plants that subsequently evolved the Kranz anatomy);
- The ratio of BS to M cells must be increased, ideally in a similar concentric organisation to Kranz anatomy;
- Enlarging and enriching BS cells with additional chloroplasts;
- Increasing the connection between M and BS cells;
- Engineering the different morphologies of the chloroplasts to mimic the morphologies of chloroplasts found in M and BS cells;
- Mirror the differing roles that M and BS cells take on in C4 photosynthesis so Rubisco reduction of CO2 occurs only in the BS cells with M cells feeding CO2 to the BS cells and excluding the oxidation of O2.
The tools we need
If we are to achieve success we still have some tools to develop and refine.
Chief among this list is a model plant that can be engineered and tested easily with speedy regeneration without requiring too much growing room. The authors point out that rice crops have some limitations in these criteria but identify Brachypodium distachyon as a model C3 plant with a small, annotated genome with quick flowering time, low growing space requirements and an efficient transformation protocol. A model such as this could hasten the engineering, testing and data gathering on conversion which can then be tested on important crop species.
A C4 model plant with similar characteristics is also required. Setaria viridis has previously been suggested as a possible model plant, as has the Fast Flowering Mini Maize.
The ability to drive and control expression of a transgene is also required. Cis-regulatory modules that promote gene expression are still under development in the wider plant synthetic biology area. This leaves a chasm between the tools we have to hand and the possibility that a large number of genes need to be differentially expressed in order to convert C3 photosynthesis to C4 photosynthesis.
Huge strides are being made with genetic manipulation, particularly with the discovery and modification of the CRISPR/Cas 9 system. But, according to the article, the maximum number of genes successfully introduced into a plant, at present, is 9. To induce C4 photosynthesis in a C3 plant, we may need the ability to stably transform a far larger number of genes plus regulatory elements, and do so without disrupting the remainder of the genome or the phenotype characteristics of our food crops.
Even when we do have these tools at the ready, we are still missing some vital information about the genes and regulatory elements that compose C4 photosynthesis. Increasing our knowledge of minutia of genetic composition and regulation of C4 systems compared to C3 systems is still a top priority. Identifying genera with the underlying predisposition that have allowed species within it to evolve from C3 to C4 for comparative analysis, particularly species displaying characteristics of a C3-C4 intermediate with sister taxa displaying C3 and C4 phenotypes, would be idyllic in assisting the study of the evolution. The authors highlight Morandia and Parthenium generas as possible true intermediates between C3 and C4 plants. Programs such as the Grass Phylogeny Working Group and the 1KP (1000 plants) project will greatly assist identifying and genotyping suitable candidates for understanding the genetics behind enhancing crop photosynthesis.
And some suggested means of pushing the research…
It is great to see that not only have the authors elucidated quite extensively the current knowledge and gaps within the field of C4 photosynthesis engineering, but have also suggested a couple of ways of advancing the research.
The first idea they suggested is synthetically replicating a simplified C4 photosynthetic system using known genetic components. The system replicates the targeting of specific enzymes to create a two-celled photosynthesis construct, limiting Rubisco to the BS cells using RNAi to interfere with its transcription in M cells. The article highlights specific transporters that can be used to transport the metabolites between the two cells.
A second suggested idea is using brute force to direct a speedy evolution of a C3 or C3-C4 intermediate species into a C4 plant. Identifying the minimum genetic requirements of a C4 plant in a candidate crop would then be followed by the repetitive growth under the selective pressure of a low CO2 atmosphere. By repeating genomic and transcription analysis of the evolving plant (if successful), a ‘mud-map’ of the road from C3 to C4 plants can be generated and be of enormous use to research seeking to synthetically install the same machinery.
Conclusion
Although its behind a pay-wall, get your hands on this article. Whether it be for a background in C4 photosynthesis or as a springboard for your own research, it is an area of immense potential that should be worthy of an X prize.
ORIGINAL: Legume Laboratory
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