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Update TRENDS in Biotechnology Vol.25 No.10 437 Engineering photorespiration in chloroplasts: a novel strategy for increasing biomass production Muhammad Sarwar Khan National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, Faisalabad, Pakistan Photosynthetic carbon metabolism is rate limiting in C3 plants because of a competing process: photorespiration. Photorespiration lowers the energy efficiency of photosynthesis by metabolizing glycolate produced by the oxygenate activity of Rubisco. The chloroplasts of Arabidopsis thaliana have recently been reported to contain a novel respiratory pathway that converts glycolate directly to glycerate and thus increases productivity by improving photosynthesis in transgenic plants. This pathway promises to widen the applicability of the approach to other C3 plants. Introduction Photosynthetic carbon metabolism is a key factor in plant growth and yield. Photosynthetic carbon fixation, catalyzed by the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is a fundamentally inefficient process for most plants; this has been extensively reviewed elsewhere [1–3]. Rubisco works very slowly; it catalyzes only a few reactions per second. A further limitation on the efficiency of carbon dioxide fixation is the ability of oxygen to bind to the active site of the enzyme in a non-productive reaction in which ribulose bisphosphate is broken down and carbon dioxide is released; this process is known as photorespiration [4]. Thus, Rubisco catalyzes two competing reactions, carboxylation and oxygenation, the rates of which depend upon the relative concentrations of CO2 and O2, as well as on temperature. Carboxylation leads to net CO2 fixation, whereas oxygenation generates glycolate that can only be metabolized outside chloroplasts by photorespiratory processes in peroxisomes and mitochondria [5]. Therefore, plant growth and yield can be improved by increased photosynthesis and/or by reduced photorespiration. The efficiency of photosynthesis in C3 plants (plants, e.g. wheat and rice, that fix CO2 in the form of three carbon-atom molecules) could be improved either if Rubisco were made more efficient or if its binding specificity for CO2 rather than for oxygen were increased. Furthermore, prospecting for more-efficient variants and concentrating CO2 in the vicinity of the enzyme by introducing the C4 pathway of photosynthesis could improve the photosynthetic efficiency of C3 plants. In C4 plants (plants, e.g. maize and sugarcane, that fix CO2 in the form of four carbon-atom molecules), enhanced CO2 fixation is caused by the coordination of two cell types: Corresponding author: Khan, M.S. ([email protected]). Available online 17 September 2007. www.sciencedirect.com mesophyll and bundle sheath cells. The arrangement of these cells around the vascular tissue, referred to as Kranz anatomy [6], differs in C4 plants, as does the mechanism of transport of metabolites between these cells [6]. Nevertheless, all C4 plants initially fix HCO3 by using phosphoenolpyruvate (PEP) carboxylase to form oxaloacetate in the cytoplasm of mesophyll cells (Figure 1a). CO2 enters mesophyll cells and is converted to HCO3 by carbonic anhydrase. Finally, the CO2 librated during decarboxylation of malate, the product of oxaloacetate, is fixed by Rubisco in the chloroplasts of neighboring bundle sheath cells [6]. However, in Hydrilla verticillata, a single-cell monocot that acclimates to declining CO2 by shifting from C3 to C4 photosynthesis [7], the librated CO2 is fixed in the same cell (Figure 1b). The discovery of an alternate version of Rubisco that would improve the efficiency of photosynthesis has long been the ‘Holy Grail’ of plant biology, but despite considerable effort, this aim has yet to be realized. Engineering C3 plants with the C4 pathway seems to be more promising, although some have questioned the benefits of concentrating CO2 in the chloroplasts of C3 plants because they are known to leak gases [8]. Recently, Kebeish et al. [9] outlined a novel approach to alleviating photorespiratory losses in Arabidopsis thaliana; this approach reduced photorespiration and enhanced photosynthesis by the release of CO2 in the vicinity of Rubisco. The approach is based on incorporating a bacterial pathway for the catabolism of the photorespiratory substrate, glycolate. Engineered chloroplastic photorespiration increases photosynthesis and biomass and thus promises to widen the applicability of this approach to C3 plants. Photorespiration and the photorespiratory pathway in C3 plants Photorespiration is the metabolism of phosphoglycolate that is produced during oxygenation catalyzed by Rubisco. Photorespiration inhibits photosynthesis by interfering with CO2 fixation catalyzed by Rubisco. Furthermore, it lowers energy efficiency by metabolizing phosphoglycolate produced by Rubisco. Energy efficiency is lowered further by the absence of a CO2-concentrating mechanism in C3 plants [4]. Overall, in photorespiratory events one molecule of CO2 is released for every two molecules of phosphoglycolate produced, a net loss of fixed carbon that reduces the production of sugars and biomass. Ammonia is also released by this reaction and needs to be ‘refixed’ by energy-consuming reactions in the chloroplasts [2]. 438 Update TRENDS in Biotechnology Vol.25 No.10 Figure 1. Schematic representation of C4 photosynthesis. (a) Photosynthesis in plants with Kranz anatomy. The CO2 enters the cytoplasm of mesophyll cell and is converted into HCO3 by carbonic anhydrase. The phosphoenolpyruvate (PEP) carboxylase fixes bicarbonate to produce oxaloacetate, which then enters the chloroplasts. The C4 acid is then converted into malate by malate dehydrogenase. The decarboxylation of malate is catalyzed by malate enzyme to generate pyruvate, thus releasing CO2 in chloroplasts of bundle sheath cells. The pyruvate diffuses back to chloroplasts of mesophyll cells, where it is converted into PEP by pyruvate orthophosphate dikinase to continue the CO2 fixation cycle. (b) Photosynthesis in a single-cell monocot, such as Hydrilla verticillata, that lacks Kranz anatomy but acclimates to declining CO2 with a shift from C3 to C4 photosynthesis. All steps involved in CO2 fixation are the same as in C4 plants; however, these steps are carried out in the same cell rather than in two different cells. Photorespiration is a seemingly wasteful reaction that adversely affects photosynthesis in C3 plants. However, it protects plants from high light intensities by playing a critical role in dissipating excess photochemical energy and thus protecting chloroplasts from over-reduction [10,11]. A variety of photorespiratory mutants of A. thaliana are able to grow in a high-CO2 environment but not at ambient CO2 concentrations, highlighting the importance of photorespiration in C3 plants [12]. Nevertheless, high CO2 levels favor photosynthesis by suppressing the photorespiratory reaction, supporting the notion that suppression of photorespiration in C3 plants could improve photosynthesis under similar environmental conditions. www.sciencedirect.com Engineering chloroplast-based photorespiration in C3 plants A pathway for the catabolism of a photorespiratory substrate, glycolate, has been reported for Escherichia coli [13,14] and plants [15]. In E. coli, glycolate dehydrogenase (GDH) uses NAD+ as an electron acceptor to oxidize glycolate to glyoxylate. However, in plants, glycolate is oxidized to glyoxylate by glycolate oxidases that use molecular oxygen in peroxisomes. Nevertheless, a glycolate pathway similar to the recently engineered photorespiratory bypass in A. thaliana was reported for Synechocystis sp. strain PCC 6803 [16]. The role of the normal photorespiration pathway in cyanobacteria is not yet clear. Update TRENDS in Biotechnology Vol.25 No.10 439 Figure 2. Engineered photorespiratory pathway in Arabidopsis thaliana. The Escherichia coli glycolate catabolic pathway is introduced to chloroplasts by step-wise incorporation of genes into the nuclear genome and post-translational targeting of proteins to the chloroplasts. The pathway works independently in chloroplasts. Carboxylation of 5-bisphosphate (RuBP) by Rubisco generates glycerate-3-phosphate for use within the Calvin cycle, whereas oxygenation generates glycolate-2-phosphate and glycerate-3-phosphate. Colored arrows and metabolites indicate the glycolate catabolic pathway in chloroplasts, whereas black arrows and metabolites in peroxisomes, mitochondria and chloroplasts depict endogenous plant photorespiration. Abbreviations: glyoxylate carboligase (GCL); glycine decarboxylase (GDC); glycolate dehydrogenase (GDH); ribulose1,5-bisphosphate (RuBP); ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); tartronic semialdehyde reductase (TSR). Taking advantage of a fundamental difference between bacterial and plant glycolate metabolic enzymes, Kebeish et al. [9] established the pathway in chloroplasts by stepwise nuclear transformation and post-translational targeting of proteins to plastids (Figure 2). Engineering the alternate photorespiratory pathway in chloroplasts was conceived based on the observation that the growth of barley and A. thaliana mutants deficient in photorespiration remained unaffected by high CO2 concentrations. However, under low CO2 concentrations the growth of mutants was drastically reduced, supporting the general perception that partial suppression of photorespiration might not be detrimental to C3 plants. To establish the glycolate catabolic pathway in chloroplasts, Kebeish et al. constructed three plasmid vectors for the transformation of Arabidopsis. The three vectors harbored five genes encoding subunits of GDH, GCL and TSR, respectively, tethered to the constitutive 35S promoter of cauliflower mosaic virus and potato-chloroplast-targeting sequence. The authors first targeted the three subunits of glycolate dehydrogenase (GDH) to chloroplasts and then introduced glyoxylate carboligase (GCL) and tartronic semialdehyde reductase (TSR) to complete the competitive photorespiratory pathway for converting glycolate to glycerate. In sequential transformations, two independent transgenic lines were produced; one (designated as the DEF line) carried genes for three subunits (D, E and F) of GDH and the other (designated as the GT line) contained genes encoding GCL and TSR. The DEF line was produced by super-transformation of a transgenic line expressing subunit F with a vector carrying genes for the D and E subunits. Crossing DEF and GT plants provided the complete pathway. Expression of the novel pathway in chloroplasts results in a reduction of photorespiratory flow and, in turn, an increase in CO2 concentration in the vicinity of Rubisco; this www.sciencedirect.com enhances carboxylation relative to oxygenation. The postillumination CO2 burst (PIB) is a measure of the CO2 released by the mitochondrial glycine decarboxylase reaction, during which glycine is converted to serine. A reduction of 30% in the measured PIB levels in DEF and GT-DEF lines in comparison to wild-type plants confirms the reduced photorespiratory flux in transgenic lines. Plant growth measurements revealed that transgenic plants expressing the glycolate pathway in their chloroplasts have a larger leaf area and an increased rosette diameter in comparison to control plants. Moreover, measurements of total fresh and dry weight showed that total plant productivity was enhanced. Interestingly, most of the described effects were also observed in plants that only overexpressed a functional GDH. However, the effects were stronger in plants overexpressing all necessary elements of the glycolate pathway. Biochemical, physiological and biophysical analyses were performed under ambient and enhanced photorespiratory conditions (low CO2 concentrations) so that the impact of the novel pathway in planta could be evaluated. The findings demonstrated that the transformants have enhanced rates of carbon fixation, growth and biomass production at low CO2 concentrations, but no differences in growth were observed in plants grown under high CO2 concentrations. Nevertheless, an increase in biomass was evident under stress conditions of high temperature or intense light, which increase photorespiration. Furthermore, increased levels of soluble sugars in plants that expressed the bacterial glycolate catabolic pathway in their chloroplasts but had no differences in Rubisco content indicate that these plants have a greater biomass. Interestingly, the reduction in the CO2 compensation point of the transgenic plants compared to wild-type plants provides further evidence for the physiological significance of the novel biochemical pathway in C3 plants. Update 440 TRENDS in Biotechnology Vol.25 No.10 Conclusion The distinguishing characteristic of the recently reported photorespiratory pathway in chloroplasts is that, unlike the endogenous photorespiratory pathway, it generates energy while eliminating the utilization of ATP and thus conserving power that is normally required to refix ammonia in the glutamine synthetase/glutamate synthase cycle. Engineered chloroplastic photorespiration increases photosynthesis and biomass and thus promises to widen applicability of the approach to other C3 crops, such as wheat, rice and particularly cotton, that are normally grown under conditions of stress. Nevertheless, further investigation into how intermediates of the novel pathway affect the basal transport system and the regulation of various metabolic processes in chloroplasts is required. Precisely how glycolate oxidation in the chloroplasts improves biomass production of field-grown crops under variable growth conditions requires further research. A possible improvement to the system is the use of regulated promoters that can induce expression of genes that encode the novel photorespiratory pathway under low-photosynthesis conditions. Another point that should be considered when transferring the pathway to C3 crops is that the plant can become loaded with several undesired marker genes; this could be avoided if the pathway were transferred into chloroplasts in the form of an operon [3]. References 1 Cleland, W.W. et al. (1998) Mechanism of Rubisco: the carbamate as general base. Chem. Rev. 98, 549–561 2 Spreitzer, P.J. (1999) Questions about the complexity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase. Photosynth. Res. 60, 29–42 3 Bock, R. and Khan, M.S. (2004) Taming plastids for a green future. Trends Biotechnol. 22, 311–318 4 Wingler, A. et al. (2000) Photorespiration: metabolic pathways and their role in stress protection. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355, 1517–1529 5 Medrano, H. et al. (1995) Improving plant production by selection for survival at low CO2 concentrations. J. Exp. Bot. 46, 1389– 1396 6 Hatch, M.D. (1992) C4 Photosynthesis: an unlikely process full of surprises. Plant Cell Physiol. 33, 333–342 7 Magnin, N.C. et al. (1997) Regulation and localization of key enzymes during the induction of Kranz-less, C4-type Photosynthesis in Hydrilla verticillata. Plant Physiol. 115, 1681–1689 8 Tolbert, N.E. (1997) The C2 oxidative photosynthetic carbon cycle. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 1–25 9 Kebeish, R. et al. (2007) Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana. Nat. Biotechnol. 25, 593–599 10 Somerville, C.R. and Ogren, W.L. (1982) Genetic modification of photorespiration. Trends Biochem. Sci. 7, 171–174 11 Somerville, C.R. (1984) The analysis of photosynthetic carbon dioxide fixation and photorespiration by mutant selection. Oxford Surveys Plant Mol. Cell Biol. 1, 103–131 12 Kozaki, A. and Takeba, G. (1996) Photorespiration protects C3 plants from photooxidation. Nature 384, 557–560 13 Lord, J.M. (1972) Glycolate oxidoreductase in Escherichia coli. Biochim. Biophys. Acta 267, 227–237 14 Pellicer, M.T. et al. (1996) The glc locus of Escherichia coli: characterization of genes encoding the subunits of glycolate oxidase and the glc regulator protein. J. Bacteriol. 178, 2051–2059 15 Leegood, R.C. et al. (1995) The regulation and control of photorespiration. J. Exp. Bot. 46, 1397–1414 16 Eisenhut, M. et al. (2006) The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in cyanobacteria. Plant Physiol. 142, 333–342 0167-7799/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2007.08.007 Free journals for developing countries The WHO and six medical journal publishers have launched the Health InterNetwork Access to Research Initiative, which enables nearly 70 of the world’s poorest countries to gain free access to biomedical literature through the internet. The science publishers, Blackwell, Elsevier, Harcourt Worldwide STM group, Wolters Kluwer International Health and Science, Springer-Verlag and John Wiley, were approached by the WHO and the British Medical Journal in 2001. Initially, more than 1500 journals were made available for free or at significantly reduced prices to universities, medical schools, and research and public institutions in developing countries. 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