Improvement of photosynthesis in rice (Oryza sativa L.) by inserting the C4 pathway
© Karki et al.; licensee Springer. 2013
Received: 9 May 2013
Accepted: 12 September 2013
Published: 28 October 2013
To boost food production for a rapidly growing global population, crop yields must significantly increase. One of the avenues being recently explored is the improvement of photosynthetic capacity by installing the C4 photosynthetic pathway into C3 crops like rice to drastically increase their yield. Crops with an enhanced photosynthetic mechanism would better utilize the solar radiation that can be translated into yield. This subsequently will help in producing more grain yield, reduce water loss and increase nitrogen use efficiency especially in hot and dry environments. This review provides a summary of the factors that need to be modified in rice so that the C4 pathway can be introduced successfully. It also discusses the differences between the C3 and C4 photosynthetic pathways in terms of anatomy, biochemistry and genetics.
KeywordsBundle sheath Chloroplast Mesophyll Photorespiration Photosynthesis
To provide adequate food and nutrition to the global population that is expected to reach 9 billion by 2050 (http://www.unpopulation.org), rice yields need to increase by at least 60% (FAO 2009). Rice is the staple food of over half of the world’s population and this rice consuming population is increasing at the rate of 1.098% per annum (http://esa.un.org/wpp/Excel-Data/population.htm). Escalating population means more demand for food, water and land at a time when the natural resource base for agriculture is being degraded because large areas of farmland are being diverted from food production to industrialization and bio-fuel production. An unpredictable climate change is threatening to further reduce agriculturally viable land due to more instances of drought and flood (http://www.fao.org/docrep/017/aq191e/aq191e.pdf). As a growing population and global climate change place increasing pressure on the world’s food supply, it is essential that we continue to improve crop performance in terms of grain productivity to keep pace with population growth. The increase in crop productivity conferred by the plant types created during green revolution period supported the population boom following the two world wars. Since then, despite the use of improved varieties and advanced technologies, the yield potential of present day rice cultivars has improved just a little indicating that these varieties have hit a yield ceiling (Akita 1994). Recently an attempt is underway to increase the rice yield potential by engineering an efficient C4 type photosynthesis into rice (Kajala et al. 2011). 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. 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. Genetic engineering provides an efficient and precise breeding tool in which only the genes of interest can be introduced even from distantly related species.
In C3 plants like rice, CO2 is assimilated into a 3-carbon compound by the photosynthetic enzyme ribulose-1, 5-bisphosphate carboxylase oxygenase (Rubisco). As the name indicates, Rubisco also catalyzes oxidation of ribulose-1, 5-bisphosphate (RuBP) in a wasteful process known as photorespiration which can incur a loss of upto 25% of previously fixed carbon (Sage 2004). At temperature above 30°C which is typical of tropical rice growing areas of the world, rate of oxygenation increases substantially and this considerably reduces the photosynthetic efficiency of C3 plants by upto 40% (Ehleringer and Monson 1993). Thus, photosynthesis of rice in the tropics and warm temperate regions becomes inefficient. The C4 plants which have CO2 concentrating mechanism within their leaves have very much reduced levels of photorespiration and thus have evolved to thrive in hot, arid environments and offer valuable insights for crop improvement strategies. Rice with a C4 photosynthesis mechanism would have increased photosynthetic efficiency while using scarce resources such as land, water, and fertilizer specifically nitrogen more effectively (Hibberd et al. 2008). Because it will perform well under high temperature as well as require less water and nitrogen, C4 rice would confer benefits on different types of rice ecosystems including the marginal lands.
C4 type photosynthesis is one of the three types of biochemical mechanisms adopted by plants to fix atmospheric CO2, others being C3 and Crassulacean acid metabolism (CAM) pathways. C4 photosynthesis has evolved more than 66 times independently (Sage et al. 2012) at least in 19 families during angiosperm evolution from C3 ancestors (Muhaidat et al. 2007) and it entails alternations of cellular structures, biochemistry and hence the development of leaves. This highly specialized form of photosynthesis essentially has developed a CO2 concentrating mechanism around the Rubisco enzyme thus eliminating the oxygenase function of Rubisco thereby reducing the wastage of energy due to photorespiration (Douce and Heldt 2000). Rubisco from C4 species is more efficient than from C3 species in terms of carboxylation (Kubien et al. 2008). 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 (Rizal et al. 2012). 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, it requires ingenuity and expertise of scientists involved in diverse disciplines such as genetic engineering, biochemistry, bioinformatics, molecular biology, photosynthesis, systems biology, physiology, plant breeding, metabolomics, etc. For the same, the C4 rice consortium was conceptualized and established which began the practical work of C4 rice engineering since 2009 (http://photosynthome.irri.org/C4rice/). This review provides an update on the requirements to develop C4 rice and progress made in the field of genetic engineering. Based on the study of the evolution of C4 from C3 species and the associated changes, the following modifications are essential to establish a functional C4 photosynthetic pathway in rice.
Increase the number and size of chloroplasts in bundle sheath cells of rice
Golden2-like (GLK) gene family members encode nuclear transcription factors that have been implicated to regulate chloroplast development in Arabidopsis, Zea mays, and the moss Physcomitrella patens (Rossini et al. 2001). In each of these 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).
Reduce the vein spacing thereby increasing the vein density in the leaf
The activity of the Calvin cycle should be significantly reduced in MC and greatly enhanced in the BSC of rice
The photorespiration in mesophyll cells has to be greatly reduced
In C3 plants carbon fixation and Calvin cycle take place in MCs. During carbon fixation, ribulose- 1, 5- bisphosphate (RuBP) - a five carbon compound, catalyzed by an enzyme ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco, EC.126.96.36.199) reacts with CO2 to form two molecules of 3-carbon compound called the 3-phosphoglycerate (3-PGA). Within the Calvin cycle, the two PGA molecules form an energy rich molecule of sugar (triose phosphate) and regenerates RuBP for the next cycle. At current atmospheric CO2 concentrations (ca. 400 ppm) Rubisco also catalyses a reaction between RuBP and O2 resulting in one molecule each of 2-phosphoglycolate and 3-PGA (Peterhansel and Maurino 2011). The 2-phosphoglycolate has to be converted back to 3-PGA through the process called photorespiration, which involves a series of biochemical reactions. During this process a loss of previously fixed carbon and nitrogen occurs and extra energy must also be used (Sharpe and Offermann 2013).
Differences in carboxylation efficiency (CE) and CO 2 compensation point (CP) between rice (C3) and sorghum (C4) at 21 and 2% oxygen level
CE (mol mol-1)
Increase in CE when O2level was reduced from 21 to 2%
CO2CP (mol mol-1)
Engineering of C4 pathway into rice
It was thought that single cell C4 system could be faster to install in C3 plants. There are attempts to engineer single cell C4 photosynthesis system in rice too (Miyao et al. 2011). To introduce single cell C4–like pathway in which MC is made to capture and release CO2 in the manner it takes place in Hydrilla verticillata (L.f) Royle., four enzymes (PEPC, PPDK, NADP-MDH, and NADP-ME) involved in the pathway were overproduced in the transgenic rice leaves (Ku et al. 1999; Fukayama et al. 2001; Tsuchida et al. 2001; Taniguchi et al. 2008). A few of the major problems encountered that need to be addressed to make a single-cell C4-like pathway in rice are: mechanism to facilitate transport activity of PEP across the chloroplast envelope, the import of OAA into the chloroplasts and the direction of the NADP-ME reaction, involvement of NADP-MDH, the presence of endogenous PEPC inside the MC chloroplast of rice and further elevation of NADP-MDH activity were reported to be necessary (Miyao et al. 2011). Terrestrial single cell C4 species such as Bienertia cycloptera, B. sinuspersici and Suaeda aralocaspica, belonging to Chenopodiaceae family also need spatial compartmentalization of the carbon assimilation and decarboxylation (Chuong et al. 2006). These species have dimorphic chloroplasts in those compartments. The earlier attempts produced futile cycle which was due to no change in anatomy, lack of appropriate transporters and the maize genes transformed into rice were not appropriately expressed in cell specific manner and were not regulated like in maize but were regulated like the endogenous rice C3 isoforms (Miyao et al. 2011).
To engineer the photosynthetic pathway from C3 to C4 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. 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 (Figure 3). 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.
The C4 photosynthetic pathway has evolved more than 66 times in different species, suggesting that C3 plants may be in some way preconditioned to C4 photosynthesis. Developing crop plants with enhanced photosynthesis will improve crop yield and make efficient use of resources in a sustainable manner. The C4 rice consortium is striving to install a maize-like photosynthetic mechanism in rice to break its yield barrier and to breed a new generation of “climate-ready” rice which will yield more even under the situations of increasing temperature and decreasing water availability. This initiative demands a holistic approach of understanding the regulatory networks and the complex mechanism underlying the C4 photosynthetic pathway and their systematic introduction into the rice genome.
We are grateful to Joan Salonga and Glenn Dimayuga for gas exchange measurements, Abigail Elmido-Mabilangan for microscopy and other staff of C4 Rice Center, International Rice Research Institute (IRRI) for their technical support. This study under the C4 Rice Center at IRRI was supported by Bill and Melinda Gates Foundation and UKAID.
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