Sep , 2021, Volume : 2 Article : 8

Sustainable Agriculture: Transforming C3 to C4 Photosynthesis

Author : Sonam S. Kale, Narendra R. Chavan, and Nilesh S. Chavan

Cite this article as:


Kale, SS., Chavan, NR.,  and  Chavan NS. (2021) Sustainable Agriculture: Transforming C3 to C4 Photosynthesis. Food and Scientific Reports. 2 (9) 40-42.


C4 photosynthesis is an evolutionary adaptation evolved in some C3 plants in flowering and both monocots and dicots. The evolution of C4 photosynthesis encompasses the recruitment of certain enzyme activities that are not present in C3 photosynthetic plants. This needs a different gene expression and enzymatic study when compared with C3 plants. Moreover, C4 plants have distinct leaf anatomy called Kranz anatomy, requiring a differential regulation of leaf development in C4. The transformation of C3 crops into C4 photosynthetic plants is a long-standing goal. However, at present, in the age of synthetic biology, C4 engineering remains a monumental task.

Keywords: C3 and C4 plants, Photosynthesis, Genome-editing, C4 engineering


It is now over half a century since the year 2016, Hal Hatch and Roger Slack first published biochemical characterization of the C4 photosynthetic pathway. To trace the fate of CO2 assimilated by sugarcane they used 14CO2 and confirmed that the first carbon compound synthesized was a C4 acidic compound i.e. Oxaloacidic acid (OAA). It results in the definition of the dicarboxylic C4 acid pathway, later called C4 photosynthesis, and the plants having this pathway were termed C4 plants. Furbank, in the year 2016, retraces these events in detail. The C4 acid pathway is not only biochemistry; rather it is a complex combination of biochemical and plant morphological characteristics. In terms of morphology, most C4 species are characterized by the Rubisco located in specialized bundle sheath cells and PEP carboxylase in the mesophyll cells. These two cells are connected by a large number of Plasmodesmata which facilitated rapid fluxes of metabolites between two cells. This is an important feature of the CO2 pump.

C4 photosynthesis pathway is now thought to be an adaptation to low atmospheric CO2 over the last 60 million years. It is recognized as one of the most dynamic processes of convergent evolution, in warm semi-arid regions (Sage et al., 2011; Sage, 2016). In his Darwin review, Sage (2016) explained the evolution of the 61 C4 families, which consist of more than 8000 species (monocots) in grasses, sedges, and eudicots, and looks at the biogeography of these species. It was also reported that the C4 pathway is related to the drought stress tolerance mechanism in B. aralocaspica which allowing it to grow in a high-temperature desert. The C4 crops like sorghum and maize play an important role in world food production and the C4 grasses such as sugarcane, miscanthus and switchgrass are the major plant sources of bioenergy. When compare with C3 crops such as rice, wheat, and potato, C4 crops have higher yields as well as increased water and nitrogen use efficiency by concentrating CO2  (Hibberd et al., 2008; Langdale, 2011). Because C4 plants approximately have 50% more photosynthetic efficiency than C3 plants. Concentrating CO2 around Rubisco located in specialized bundle sheath cells significantly reduces the oxygenation reaction. An increase in the photosynthetic performance is a promising strategy to overcome low yield and might provide the foundation of a second Green Revolution. Hence engineering C4 features into C3 plants is important for the enhancement of crop yield.

 Avenues for transforming C3 into C4 plants

Genome and gene regulation

As compared to traditional methods of crop improvement the increase in crop productivity must be attempted by using novel techniques and tools. As this is a very complex trait, a full molecular knowledge of the metabolic and anatomical adaptations will be necessary to engineer this photosynthetic trait. With the development of high-throughput NGS technologies, the genomes of crop plants have been sequenced and also annotated completely. The crop plants such as rice (Oryza sativa), sorghum (Sorghum bicolor), maize (Zea mays) for which all the genomics information is available and make it possible to reconstruct the genome-scale metabolic network of the plants. Flux Balance Analysis (FBA) is a suitable method to analyze a network of metabolic pathways without demanding detail kinetic parameters. Huang et al., (2017) developed a comparative genomics-based cross-species genome scanning approach for mining of genes in C4 evolution under positive selection. This is independent of knowledge of the biochemical pathways involved. With the availability of genetics and genomics (structural and functional genomics) approaches which are being used in evolutionary and biochemical research perspectives to answer questions in C4 photosynthesis research.

Advance technologies

In the recent past, C4 photosynthesis made use of important physiological techniques viz., exchange of gas measurement in plants resulted in the development of unique gas exchange feature of CO2 assimilation rate in C4 plants. This led to a good understanding of C4 plants respond to environmental variables such as CO2, light, and temperature. The first-ever model for C4 photosynthetic gas exchange based on biochemical studies has been correctly predicted its CO2 assimilation function, with first estimates of bundle sheath CO2 partial pressures. This functional model allows the link between gas exchange and leaf biochemistry has become an essential tool. Bellasio (2017) has generated a stoichiometric model for C2, C2+ C4, C3, and C4 pathways in which different decarboxylating enzymes, metabolite traffic, and energetics are explicitly included. 14C pulse-chase techniques used by Hatch and Slack to unravel the mysteries of C4 photosynthesis have been replaced by mass spectrometric-based measurements of 13CO2 labeling kinetics, provide a wealth of information compared to past experiments. Arrivault et al., (2017) used this technique first time in Maize to establish pool sizes and metabolites gradients using cell type fractionation. However, efforts to introduce C4 traits into C3 plants have been hampered by an incomplete gene annotation required to support the trait (Weber and Bar-Even, 2019). Hence, the identification and functional validation of the associated genes seem to be a fundamental challenge faced by researchers and a crucial step toward the introduction of the C4 pathway into important C3 crop plants.

Based on the available knowledge on C4 photosynthesis, the following engineering steps would be necessary to acquire it in C3 photosynthetic plants. 1) Increased vein density by the introduction of higher-order veins in young leaf primordia.  2) Increase in bundle sheath cell to mesophyll cell ratio and concentric organization of the cells. 3)  Bundle sheath cell morphology adaptation in plants. In C4 plant these cells are enlarged and contain more chloroplast compared to C3 plants. Additionally, they are interconnected with mesophyll cells which results in high metabolite flux between these two cell types by plasmodesmata. 4)   Introduction of dimorphic chloroplasts in BS and M cells. Chloroplasts occur in two different forms: M cell chloroplasts do not contain starch grains. Whereas BS chloroplasts accumulate starch and are distributed. The engineering of two different types of chloroplast can increases photosynthetic efficiency.

 Recent advances in C4 research

With the development of high-throughput sequencing technologies (NGS) and C4 research to date gain new momentum. Systems biology based on transcriptomics data helped to identify unknown components of C4 pathways and provided molecular insight in the understanding about the development of the kranz anatomy. Transcriptomics not only gives information about the candidate genes involved in C4 trait development but also provides information on C4 evolution that might be useful for the engineering of C4 plants.

The C4 plant engineering relies on genome editing technologies and in planta gene targeting. The technologies including CRISPR/Cas9, used for inducing the desired changes in plants, RNA interference which triggers mutations of targeted loci substituting gene silencing, and gene targeting based on homologous recombination. Engineering of C4 plants might not only require the transfer of new additional genes, it will likely require the knockout of genes. The idea comes from functional genomics/transcriptomics studies which revealed that many genes were completely down regulated in the C4 photosystem when compared with C3. CRISPR/Cas9 would be a knocking-out technology for repressing C3 features e.g. deep lobing of chlorenchyma cells in rice.

To modify the C3 features, the maximum number of genes introduced was nine which lead to successful engineering. Whereas, the predicted gene number needed for successful engineering of a complete C4 pathway with optimized leaf anatomy will exceed this number. However, genome landing pads will be required to enable the transformation of multigene constructs into the plants to avoid undesirable changes. Some cyanobacteria and plant species have carbon concentration mechanisms (CCMs) to increase the carboxylase activity of RuBisCO and thus limit their rate of photorespiration known as C4 and CAM photosynthesis. Hence it is synthetic biology key driver to introduce CCMs into C3 plants aimed at improving photosynthetic capabilities of C3 plants.


Photosynthesis is an engine for life on earth that has high engineering potential by using applied genomics technologies. Converting C3 into C4 is a way to increases the photosynthetic efficiency and ultimately the yield of a crop plant to fulfill the present and future food demand. Recent developments and advancements in genome editing have been used to boost the photosynthetic ability for sustainability. Moreover, even though more targeted and precise genetic manipulation strategies have been recently generated in plant photosynthesis, a range of challenges remain. 


Arrivault, S., Obata, T., Szecowka, M., Mengin, V., Guenther, M., Hoehne, M., Fernie, A. R., & Stitt, M. (2017). Metabolite pools and carbon flow during C4 photosynthesis in maize: 13CO2 labeling kinetics and cell type fractionation. Journal of experimental botany68(2), 283–298.

Bellasio C. (2017). A generalized stoichiometric model of C3, C2, C2+C4, and C4 photosynthetic metabolism. Journal of experimental botany68(2), 269–282.

Furbank R. T. (2016). Walking the C4 pathway: past, present, and future. Journal of experimental botany67(14), 4057–4066.

Hibberd, J. M., Sheehy, J. E., & Langdale, J. A. (2008). Using C4 photosynthesis to increase the yield of rice-rationale and feasibility. Current opinion in plant biology11(2), 228–231.

Huang, P., Studer, A. J., Schnable, J. C., Kellogg, E. A., & Brutnell, T. P. (2017). Cross species selection scans identify components of C4 photosynthesis in the grasses. Journal of experimental botany68(2), 127–135.

Langdale J. A. (2011). C4 cycles: past, present, and future research on C4 photosynthesis. The Plant cell23(11), 3879–3892.

 Sage R. F. (2017). A portrait of the C4 photosynthetic family on the 50th anniversary of its discovery: species number, evolutionary lineages, and Hall of Fame. Journal of experimental botany68(2), 4039–4056.

Sage, R. F., Christin, P. A., & Edwards, E. J. (2011). The C (4) plant lineages of planet Earth. Journal of experimental botany62(9), 3155–3169.

Weber, A., & Bar-Even, A. (2019). Update: Improving the Efficiency of Photosynthetic Carbon Reactions. Plant physiology179(3), 803–812.



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