Insertion of an Efficient C4 Type Photosynthetic Pathway in Rice: An Overview
Authors: Shailesh Kumar*1, Neelambri1, A K Singh1, Sanam Kumari1 , Sweta Mishra2, Shika Kumari1
1 DRPCAU, Pusa, Samastipur, Bihar ;
2 SDAU, S K Nagar, Gujarat

The grain productions of crop plants were largely improved during green revolution due to maximization of light interception and harvest index. However, RUE (efficiency with which intercepted PAR is converted into dry matter) also decide the yield potential of crop plants which remain unexploited during green revolution and it can act as a potential source for significant new genetic improvement. As per theoretical models prediction, insertion of C4 type photosynthetic pathway in C3 crop plants would improve RUE of C3 crops by approximately 50%. This led to the suggestion that converting crops from C3 to C4 could mitigate the global food crisis. Recently an effort is in progress to increase the rice yield potential by engineering an efficient and functional C4 type photosynthesis into rice.


The grain productions of crop plants were largely improved during green revolution due to maximization of light interception and harvest index. However, in current years, plant biologist has unsuccessful to systematically increase yields in line with increasing population. Yield potential of crop plants is decided by of four major factors: (a) total incident solar radiation accrued over the growing season, (b) efficiency of the plant to intercept photosynthetically active radiation (PAR), (c) efficiency with which intercepted PAR is converted into dry matter (radiation use efficiency, RUE) and (d) amount of resources partitioned to the grain (harvest index). Maximization of light interception and harvest index of crop plants led to the Green Revolution. Extending the growing season is unwelcome move, because management practices will have to be optimized. This leaves RUE as a potential source for significant new genetic improvement. Theoretical models predict RUE of C3 crops would be improved by approximately 50% by using C4 photosynthesis. This led to the suggestion that converting crops from C3 to C4 could mitigate the global food crisis (Covshoff et al. 2012). Recently an attempt is underway to increase the rice yield potential by engineering an efficient C4 type photosynthesis into rice. For this, a set of genes which regulate leaf anatomy and biochemical processes have to be inserted into rice and expressed in an appropriate manner which is currently not possible solely by conventional plant breeding techniques. This novel approach to modify the photosynthesis system of rice is a challenging and long term endeavor because the C4 pathway is very complex and many factors controlling the mechanism are still unknown. Therefore, genetic engineering to improve the photosynthetic pathway of rice would provide sufficient opportunity to enhance the actual grain productivity as well as the yield potential. For the same, the C4 rice consortium was conceptualized and established which began the practical work of C4 rice engineering since 2009. A coordinated international effort to introduce this ability into rice has already produced exciting results (Caemmerer et al.2012).

C4 type photosynthesis

Apart from C3 and Crassulacean acid metabolism (CAM) pathways, C4 type photosynthesis is also adopted by several C4 plants to fix atmospheric CO2. The biochemical mechanism of C4 photosynthesis has evolved more than 66 times independently in many species during angiosperm evolution from C3 ancestors and it entails alternations of cellular structures, biochemistry and hence the development of leaves. To eliminate the oxygenase function of Rubisco enzyme the C4 photosynthetic pathways/system has essentially developed a CO2 concentrating mechanism around the Rubisco enzyme and reducing the wastage of energy due to photorespiration. Rubisco from C4 species is more efficient than from C3 species in terms of carboxylation. The other associated benefits of the C4 system include higher water use efficiency because steeper concentration gradient for CO2 diffusion can be maintained through partly closed stomata, higher radiation use efficiency as C4 photosynthesis efficiency does not get saturated at high light intensity (Rizal et al. 2012) and higher nitrogen use efficiencies because it will require less Rubisco and hence less nitrogen. C4 plants are potentially more productive at higher temperatures typically experienced by rice. To take advantage of this more efficient photosynthetic system at a time when the population and food prices are soaring, there are efforts towards inserting the C4 mechanism such as that found in maize into rice (Karki et al. 2013).

Gene identification to install C4 photosynthesis in rice and current progress

The detailed evolution study of C4 from C3 species necessitated the few important modification to establish a efficient and functional C4 photosynthetic pathways in rice (Karki et al. 2013).

(a) Increase the number and size of chloroplasts in bundle sheath cells of rice (b) Reduce the vein spacing thereby increasing the vein density in the leaf (c) The activity of the Calvin cycle should be significantly reduced in MC (mesophyll cells) and greatly enhanced in the BSC (bundle sheath cells) of rice (d) The photorespiration in mesophyll cells has to be greatly reduced (e) Engineering of C4 pathway into rice. The nuclear transcription factors encoded by gene family Golden2-like (GLK) have been found to regulate chloroplast development in Arabidopsis, Zea mays, and the moss Physcomitrella patens (Rossini et al. 2001). In all above mentioned species, GLK genes exist as a homologous pair named as GLK1 and GLK2 (Waters et al. 2009). In moss and Arabidopsis the GLK genes are redundant and functionally equivalent whereas in maize and sorghum GLK genes act in a cell-type-specific manner to direct the development of dimorphic chloroplasts (Waters et al. 2008; Wang et al. 2013a). In maize, Golden2 (G2) and its homologue ZmGLK1 transcripts accumulate primarily in BS and M cells, respectively, suggesting a specific role for each gene regulating the dimorphic chloroplast differentiation (Wang et al. 2013a). Therefore incorporation of Golden2 (G2) gene may help in development of dimorphic chloroplasts in rice. It is also reported that the for Kranz anatomy patterning, SCARECROW/ SHORTROOT regulatory network has been determined to be one of the important components. The leaves of C3 plants with mutated Scarecrow gene was normal, while in the C4 plants mutation in the same gene damaged the Kranz anatomy (Slewinski et al. 2012; Wang et al. 2013b). Recently, it has been shown that introduction of maize chromosomes into oat could increase the BSC size and reduce vein spacing in C3 oat leaves demonstrating that the anatomy of C3 leaf can be modified (Tolley et al. 2012). Moreover, a large effort has been put to screen sorghum (C4) mutants with increased vein spacing and rice (C3) mutants with reduced vein spacing so that the genes controlling vein spacing trait can be identified (Rizal et al. 2012). To make functioning C4 rice, Rubisco activity has to be greatly reduced in MCs and increased in BSCs which then confines the Calvin cycle to the BSCs of rice, like in a C4 system. On the other hand, genes encoding several C4 enzymes such as β carbonic anhydrase (CA) and PEPC have to be over expressed in cytosol of MCs of rice in order to facilitate the primary CO2 fixation so that CO2 can be concentrated and supplied to Rubisco in the BSCs. The C4 cycle also involves extensive transport of metabolites across the chloroplast envelope membrane and plasmalemma of MC and BSC. As such, in addition to the core C4 enzymes namely CA, PEPC, pyruvate orthophosphate (Pi) dikinase (PPDK, EC, NADP dependent malate dehydrogenase (NADP-MDH, EC and NADP-dependent malic enzyme (NADPME, EC, C4 pathway also requires insertion of metabolite transporters for oxaloacetate, malate, triose-phosphate and pyruvate into rice to provide increased transport capacity for the C4 cycle intermediates so that the Calvin cycle can function effectively in the BSCs (Weber and von Caemmerer 2010). C4 genes such as CA, PEPC, PPDK, NADP-ME, and NADP-MDH are cloned from maize and transformed into rice. Also the transporters that were over expressed in the C4 metabolic pathways such as 2-oxoglutarate /malate transporter (OMT1), dicarboxylate transporter1 (DiT1), dicarboxylate transporter2 (DiT2), PEP/phosphate transporter (PPT1), mesophyll envelope protein (MEP) and triose-phosphate phosphate translocator (TPT) that were recently identified through proteomics of maize BS and MS cells (Friso et al. 2010) are being transformed into rice.

The insertion of functional and efficient C4 photosynthetic pathway in C3 rice within two decades, which took million of years in nature, C4 rice consortium began the simultaneous gene discovery and engineering of already known genes into rice aiming to form C4 rice with Kranz type anatomy. The C4 rice consortium members are also involved in discovering novel genes related to Kranz anatomy (Wang et al. 2013b). Once tested, the promising candidate genes controlling the Kranz anatomy will also be introduced in the rice plants that have been engineered with the C4 biochemical pathway genes (Karki et al. 2013).


The enhancement of photosynthetic capacity of C3 plant through incorporation of C4 type pathway will certainly improve crop yield and make efficient use of water and nitrogen in a sustainable manner. It will help in development of new generation of “climate-smart” rice. Certainly a holistic approach is needed to understand the complex regulatory mechanism associated with C4 photosynthetic pathways and their systemic incorporation into rice genome.

1. Wang P, Kelly S, Fouracre JP, Langdale JA (2013b) Genome-wide transcript analysis of early maize leaf development reveals gene cohorts associated with the differentiation of C4 Kranz anatomy. Plant J 75: 656-670.

2. Rizal G, Karki S, Thakur V, Chatterjee J, Coe RA, Wanchana S, Quick WP (2012) Towards a C4 rice. Asian J Cell Biol 7:13-31.

3. Karki S, Rizal G, Quick WP (2013) Improvement of photosynthesis in rice (Oryza sativa L.) by inserting the C4 pathway. Rice 6: 2-8.

4. Covshoff S and Hibberd J M (2012) Integrating C4 photosynthesis into C3 crops to increase yield potential. Current Opinion in Biotechnology 23:209-214.

5. Caemmerer SV, Quick WP, Furbank RT (2012) The Development of C4 Rice: Current Progress and Future Challenges. Science 29 (336) : 1671-1672.
6. Friso G, Majeran W, Huang MS, Sun Q, van Wijk KJ (2010) Reconstruction of metabolic pathways, protein expression, and homeostasis machineries across maize bundle sheath and mesophyll chloroplasts: large-scale quantitive proteomics using the first maize genome assembly. Plant Physiol 152:1219-1250

7. Weber APM, von Caemmerer S (2010) Plastid transport and metabolism of C3 and C4 plants: comparative analysis and possible biotechnological exploitation. Curr Opin Plant Biol 13:257-265.

8. Tolley BJ, Sage TL, Langdale JA, Hibberd JM (2012) Individual maize chromosomes in the C3 plant oat can increase bundle sheath cell size and vein density. Plant Physiol 159:1418-1427.

9. Wang P, Fouracre J, Kelly S, Karki S, Gowik U, Aubry S, Shaw MK, Westhoff P, Slamet-Loedin IH, Quick WP, Hibberd JM, Langdale JA (2013a) Evolution of GOLDEN2-LIKE gene function in C3 and C4 plants. Planta 237:481-495.

10. Slewinski TL, Anderson AA, Zhang C, Turgeon R (2012) Scarecrow plays a role in establishing Kranz anatomy in maize leaves. Plant Cell Physiol 53:2030-2037.

About Author / Additional Info:
Interested in photosynthesis research