Photosynthetic organisms became terrestrial around 450 million years ago, and over these years land plant morphology and photosynthetic mechanisms have diversified (Sage, 2004). There are three basic photosynthetic pathways used by plants. In C3 plants, CO2 is fixed by Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) in the Calvin cycle to generate a three-carbon compound. Plants that use the C4 and Crassulacean Acid Metabolism (CAM) pathways are evolved from C3 plants, and in both cases, a four-carbon organic acid is initially formed from fixation of HCO3- . The efficiency with which CO2 is supplied to RuBisCO is increased through the addition of this C4 carbon shuttle. Rubisco, the primary CO2-fixing enzyme in C3 plants, is a poor catalyst of CO2 at current atmospheric conditions. It has a tendency of using CO2 as well as O2 as substrate. Rubisco's oxygenase activity requires the recycling of phosphoglycolate in the photorespiratory pathway, resulting in an energy cost and loss of previously fixed CO2. C4 plants, taking advantage of the Kranz anatomy, compartmentalize photosynthetic reactions between mesophyll (M) and bundlesheath (BS) cells. Mesophyll cells surround enlarged BS cells which are arranged in concentric circles around veins, giving rise to Kranz anatomy. C4 acids generated in mesophyll cells diffuse to the closely spaced BS cells, where the Calvin cycle operates. Carbonic anhydrase (CA), phosphoenolpyruvate carboxylase (PEPC), NADP-malate dehydrogenease (MDH), pyruvate orthophosphate dikinase (PPDK) and the related regulatory proteins accumulate in mesophyll cells, whereas NADP-malic enzyme (ME) and RuBisCO are restricted to the BS. Genes encoding all of these enzymes are present in C3 plants, but expression levels are much lower than in C4 species.
C4 plants have 50% higher radiation use efficiency than C3 plants, due to differences in photosynthesis. In comparison to C3 crops, C4 crops have better yield and increased water and nitrogen use efficiency. As the world population is increasing at alarming rates, the need for another "green revolution" is impending to meet demands for food. In C3 crop like rice, yield is source limited, ie, the photosynthetic activity of leaves are inadequate to fill the larger number of florets. One probable solution to this problem is to establish an efficient, higher capacity photosynthetic mechanism in rice, the C4 pathway. With the ambitious aim of "C4 rice", an international C4 Rice Consortium led by the International Rice Research Institute (IRRI, Philippines) with 24 participating research groups, involving scientists from both advanced institutions and the developing countries was set up in 2008 . Many projects are underway within the C4 Rice Consortium, including physiological phenotyping, screening of rice mutants for characteristics of Kranz anatomy and to introduce the genes of C4 photosynthesis into rice (Covshoff et al. 2012).
Most important aspect is identifying and transforming the genes necessary to install C4 photosynthesis in rice. The functional C4 cycle also requires down-regulation of part of the Calvin-Benson cycle in mesophyll cells. Another aspect of of C4 engineering is addition of the transporters required to support ﬂuxes of metabolites between subcellular compartments of the C4 cycle. Candidate transporters identiﬁed from differential proteomic studies (Friso et al. 2010) by as well as appropriate cell type-speciﬁc accumulation in maize are cloned from maize, which include a putative OAA/malate antiporter (OMT1), putative dicarboxylate transporters (DiT1 and DiT2), and the PEP/ phosphate translocator (PPT1). Mesophyll speciﬁc expression patterns of maize promoters in rice have been characterized for ZmPEPC and ZmPPDK, and these promoters have been successfully used to drive strong M-speciﬁc expression in rice. Transgenic plants with proper localization and appropriate functional levels of each protein will be crossed to stack the transgenes. Subsequently, C4 photosynthesis can be transferred to elite rice varieties with better agronomic attributes, by conventional breeding.
Though the specialized leaf anatomy is integral to two-celled C4 photosynthesis, none of the genes controlling C4 leaf anatomy have been identified. Similarly genes related to suberization of BS cells, plasmodesmatal connectivity between M and BS cells, and production and positioning of dimorphic chloroplasts are yet to be characterized (Kajala et al. 2011). The leaves of C4 plants exhibit increased vein density, which decreases the diffusion distance of C4 acids from M to BS cells and the transport distance of the photosynthate into the vasculature. Activation-tagged rice populations are being screened for decreased vein spacing and C4-like leaf anatomical characteristics. Similarly, mutant populations of Sorghum bicolor and Setaria viridis were generated to study the loss of C4 characteristics (Brutnell et al. 2010, Kajala et al. 2011). Rice mutants with decreased vein density and an increasing number of M cells between veins have been successfully identified. Combining the information from screens of mutagenized C4 Sorghum bicolor and Setaria viridis and activation-tagged rice populations might help to identify candidate genes in the C3-to-C4 switch. The efficiency of C4 plants is determined by alterations in leaf chlorophyll and a/b ratio hence, the activation-tagged rice mutants are screened for changes in chlorophyll content. It is expected that a C4 rice prototype might be available within 3 years. However, another 15 years of research are required for optimization of the phenotype and field testing for C4 rice to become ready for cultivation in farmers' fields (Caemmerer et al. 2012).
Sage, RF. "The evolution of C4 photosynthesis". New Phytologist 2004, 161:341-370
Covshoff, Sarah, and Julian M. Hibberd. "Integrating C4 photosynthesis into C3 crops to increase yield potential." Current Opinion in Biotechnology 23, 2 (2012): 209-214
Brutnell, Thomas P., Lin Wang, Kerry Swartwood, Alexander Goldschmidt, David Jackson, Xin-Guang Zhu, Elizabeth Kellogg, and Joyce Van Eck. "Setaria viridis: a model for C4 photosynthesis." The Plant Cell Online 22, 8 (2010): 2537-2544.
Kajala K, Covshoff S, Karki S, Woodfield H, Tolley BJ, Dionora MJ, Mogul RT, Mabilangan AE, Danila FR, Hibberd JM, Quick W P. "Strategies for engineering a two-celled C4 photosynthetic pathway into rice." Journal of experimental botany 62, 9 (2011): 3001-3010.
von Caemmerer, Susanne, W. Paul Quick, and Robert T. Furbank. "The development of C4 rice: current progress and future challenges." Science 336, 6089 (2012): 1671-1672.
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