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P1: FUM March 30, 2001 18:0 Annual Reviews AR129-11 Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001. 52:297–314 c 2001 by Annual Reviews. All rights reserved Copyright ° MOLECULAR ENGINEERING OF C4 PHOTOSYNTHESIS Makoto Matsuoka BioScience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan; e-mail: [email protected] Robert T Furbank CSIRO Plant Industry, G.P.O. Box 1600, Canberra ACT 2601, Australia; e-mail: [email protected] Hiroshi Fukayama and Mitsue Miyao Laboratory of Photosynthesis, National Institute of Agrobiological Resources, Kannondai, Tsukuba 305-8602, Japan; e-mail: [email protected] Key Words C3 plants, C4 plants, carbon metabolism, evolution, transgenic plants ■ Abstract The majority of terrestrial plants, including many important crops such as rice, wheat, soybean, and potato, are classified as C3 plants that assimilate atmospheric CO2 directly through the C3 photosynthetic pathway. C4 plants such as maize and sugarcane evolved from C3 plants, acquiring the C4 photosynthetic pathway to achieve high photosynthetic performance and high water- and nitrogen-use efficiencies. The recent application of recombinant DNA technology has made considerable progress in the molecular engineering of C4 photosynthesis over the past several years. It has deepened our understanding of the mechanism of C4 photosynthesis and provided valuable information as to the evolution of the C4 photosynthetic genes. It also has enabled us to express enzymes involved in the C4 pathway at high levels and in desired locations in the leaves of C3 plants for engineering of primary carbon metabolism. CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . METABOLIC ENGINEERING OF C4 ENZYMES IN C4 PLANTS . . . . . . . . . . . . Rate-Limiting Reactions in C4 Photosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . Transgenic C4 Plants and the CO2 Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MOLECULAR ENGINEERING OF C4 ENZYMES IN C3 PLANTS . . . . . . . . . . . How to Overproduce C4 Enzymes in the Leaves of C3 Plants . . . . . . . . . . . . . . . . 1040-2519/01/0601-0297$14.00 298 300 300 302 303 303 297 P1: FUM March 30, 2001 298 18:0 Annual Reviews AR129-11 MATSUOKA ET AL Physiological Impacts of Overproduction of C4 Enzymes in C3 Plants . . . . . . . . . 307 FUTURE PERSPECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310 INTRODUCTION The majority of terrestrial plants, including many important crops such as rice, wheat, barley, soybean, and potato, assimilate atmospheric CO2 directly through the C3 photosynthetic pathway, also known as the Calvin cycle, and these are classified as C3 plants. The enzyme of primary CO2 fixation in this pathway, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), reacts not only with CO2 but also with O2, leading to photorespiration, which essentially wastes assimilated carbon. Under current atmospheric conditions, potential photosynthesis in C3 plants is suppressed by oxygen by as much as 40%. The extent of suppression further increases under stress conditions such as drought, high light, and high temperature, through a decline of the CO2 concentration inside leaves due to closure of stomata. C4 plants such as maize, sorghum, and sugarcane have evolved a novel biochemical mechanism to overcome photorespiration. In addition to the C3 pathway, they use the C4 photosynthetic cycle to elevate the CO2 concentration at the site of Rubisco and thus suppress its oxygenase activity. This mechanism enables C4 plants to achieve elevated photosynthetic capacity particularly at higher temperatures, of up to twice as high as that of C3 plants, in addition to higher waterand nitrogen-use efficiencies (for reviews of C4 photosynthesis, see 17, 22, 40a). Consequently, it has long been postulated that the transfer of C4 traits to C3 plants could improve the photosynthetic performance of C3 species. Leaves of C4 plants have two types of photosynthetic cells, the mesophyll cell (MC) and bundle sheath cell (BSC). While all the photosynthetic enzymes are confined in MCs in C3 plants, they are localized in MCs and/or BSCs in C4 plants (Table 1). In addition, C4 plants show extensive venation, with a ring of BSCs surrounding each vein and an outer ring of MCs surrounding the bundle sheath. This unique leaf structure, known as Kranz anatomy, and the cell-specific compartmentalization of enzymes are essential for operation of the C4 pathway. The initial fixation of CO2 in the C4 pathway occurs in the MC cytosol by phosphoenolpyruvate carboxylase (PEPC) to form the C4 acid oxaloacetate (OAA). OAA is either reduced to malate by NADP-malate dehydrogenase (NADP-MDH) or transaminated to aspartate by aspartate aminotransferase (AspAT). The resultant C4 acid is transported to the BSCs and then decarboxylated to release CO2 in the vicinity of Rubisco. The decarboxylation reaction is catalyzed by one or more of the three enzymes, namely, NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PEP-CK), and C4 plants are classified into three subtypes depending on the major decarboxylation enzyme. A C3 acid generated by the decarboxylation is shuttled back to MCs to regenerate the primary CO2 acceptor phosphoenolpyruvate (PEP) by pyruvate,orthophosphate dikinase (PPDK) in the MC chloroplasts. P1: FUM March 30, 2001 18:0 Annual Reviews AR129-11 ENGINEERING OF C4 PHOTOSYNTHESIS 299 TABLE 1 Location of major photosynthetic enzymes in leaves of C4 plants Enzyme Gene C4 subtype(s) Location Function Rubisco (C3) rbc All BSC, Chlt Net CO2 fixation PEPC (C4) Ppc All MC, Cyt Initial CO2 (HCO3−) fixation PPDK (C4) Pdk All MC, Chlt Regeneration of PEP NADP-MDH (C4) mdh NADP-ME MC, Chlt OAA → malate NADP-ME (C4) Me NADP-ME BSC, Chlt Decarboxylation of malate NAD-ME (C4) NAD-ME (PEP-CK) BSC, Mit Decarboxylation of malate AspAT (C4) NAD-ME PEP-CK MC, Cyt; BSC, Mit OAA → asparate (MC) Asparate → OAA (BSC) PEP-CK (C4) PEP-CK (NADP-ME) BSC, Cyt Decarboxylation of OAA CA (C4) All MC, Cyt CO2 → HCO3− Enzymes mentioned in this article are listed. MC, mesophyll cells; BSC, bundle sheath cells; Cyt, cytosol; Chlt, chloroplasts; Mit, mitochondria. The enzymes involved in the C4 pathway (C4 enzymes) had previously been considered to be specific for C4 plants, since the activities of homologues are low in C3 plants and their kinetic properties are usually different from those of C4 enzymes. However, recent comparative studies have revealed that C3 plants have at least two different types of genes, one encoding enzymes of “housekeeping” function and the other very similar to the C4 photosynthetic genes in C4 plants, although expression of the latter is very low or even undetectable in C3 plants. Based on this finding, it is postulated that the C4-specific genes evolved from a set of preexisting counterpart genes in ancestral C3 plants, with some modifications in expression patterns in leaves and kinetic properties of enzymes (33, 40). The recent application of recombinant DNA technology to plant metabolism has considerably advanced our understanding of the regulation of photosynthesis (18). By altering the levels and properties of key enzymes in photosynthesis in transgenic C4 plants, we are now able to deepen our understanding of the mechanism of C4 photosynthesis. Cell-specific expression of C4 photosynthetic genes can also be probed using reporter gene constructs in transgenic plants (9, 33). Our understanding of the evolution of photosynthetic carbon metabolism can be advanced by examining the consequences of the transfer of C4-specific genes to transgenic C3 plants. Lastly, a great challenge is to use these techniques and C4 genes to alter carbon metabolism of C3 crop plants to answer fundamental questions concerning agronomic performance and to improve crop productivity. Here we review progress in these key areas of C4 photosynthesis research. P1: FUM March 30, 2001 300 18:0 Annual Reviews AR129-11 MATSUOKA ET AL METABOLIC ENGINEERING OF C4 ENZYMES IN C4 PLANTS The capacity to alter photosynthesis in transgenic plants with a high degree of precision has provided a powerful tool for studying rate-limiting processes in photosynthesis and carbon partitioning in vivo (see 18). This technology, using the C4 dicot Flaveria bidentis (8) along with the isolation of C4 mutants of Amaranthus edulis requiring high CO2 for growth (see 10), has allowed the analysis of control points in the C4 pathway in intact plants. In this section we review the contribution of these approaches to our understanding of C4 metabolism and mechanism. Rate-Limiting Reactions in C4 Photosynthesis One of the great challenges in plant biochemistry has been to transfer the vast amount of knowledge on enzyme kinetics in vitro to the control of flux through metabolic pathways in vivo. Using antisense, cosuppression, ectopic overexpression, and the mutational approach described above, we now have a relatively clear picture of the role individual enzymes play in controlling photosynthetic flux in C4 plants. Table 2 summarizes this work by presenting control coefficients (Cj) for the individual enzymes examined so far. These coefficients were mostly determined at saturating light and ambient CO2. The significance of control coefficients has been reviewed elsewhere (1) but a Cj of one indicates that flux is fully controlled at this step in the pathway, whereas a value of zero indicates no control over flux. The results summarized in Table 2 indicate that the bulk of control in C4 photosynthesis lies with the three enzymes Rubisco, PPDK, and PEPC. Experiments where expression of the gene encoding the small subunit of Rubisco (rbcS) was reduced by antisense in Flaveria indicate that at high light, a control coefficient of 0.5 to 0.6 is likely for this enzyme (15, 16). This result was somewhat surprising as it has been suggested that the high CO2 concentration in BSCs has allowed C4 plants to reduce the amount of Rubisco in BSCs, relative to that in C3 plants (one TABLE 2 Control coefficients (Cj) for photosynthetic enzymes in C4 plants determined by measuring CO2 assimilation under saturating illumination and ambient CO2 Enzyme C4 species Technique Cj References Rubisco F. bidentis Antisense RNA 0.5–0.6 15, 16 PPDK F. bidentis Antisense RNA 0.2–0.4 15 PEPC A. edulis Mutation 0.35 2, 10 NAD-ME A. edulis Mutation ∼0 11 NADP-MDH F. bidentis Cosuppression ∼0 47 P1: FUM March 30, 2001 18:0 Annual Reviews AR129-11 ENGINEERING OF C4 PHOTOSYNTHESIS 301 third to one quarter of C3 levels), without affecting photosynthesis, resulting in greater nitrogen-use efficiency (41). It appears that this evolutionary optimization of Rubisco level has poised nitrogen investment in this protein to the minimum effective level in wild-type plants. As with C3 plants, it would be instructive to elevate Rubisco levels in transgenic C4 plants; however, the overexpression of the chloroplast-encoded large subunit of Rubisco currently provides a technical barrier to these experiments. Control of photosynthesis by PPDK and PEPC is more difficult to assess. Both these enzymes are subject to complex regulation by reversible protein phosphorylation and in the case of PEPC, mutants have been shown to increase the enzyme activation state in response to decreased enzyme levels (10). In the case of PEPC, a control coefficient of 0.35 was calculated for Amaranthus mutants at saturating light and ambient CO2 (2). A similar value was found for PPDK in transgenic Flaveria containing an antisense gene construct targeted to this enzyme (15). The fact that the Cj values for these three enzymes sum to greater than one may be due to the different growth conditions used in each case or possibly interspecific or decarboxylation-type variations in enzyme levels. Two of the enzymes in C4 photosynthesis examined thus far appear to exert little or no control over photosynthetic flux in vivo: NADP-MDH (47) and NADME (11). In the case of NADP-MDH, transgenic Flaveria containing antisense or cosuppression constructs showed no effect of reduced enzyme level until plants contained less than 10% of wild-type levels of the protein (47). Even after it is taken into account that these plants showed increased enzyme activation state to compensate for reduced enzyme levels, Cj for this thioredoxin-regulated enzyme was effectively zero under all conditions examined (47). Similarly, in heterozygous Amaranthus mutants with an approximately 50% reduction in NAD-ME activity, no effect on photosynthesis could be detected, indicating that this enzyme effectively exerts no control over photosynthetic flux (11). Although the experiments described above in which high control coefficients are observed indicate where control of flux in C4 photosynthesis lies, the observations that several “key enzymes” appear to have no regulatory role are equally intriguing. NADP-MDH is highly regulated by light through the thioredoxin-mediated reduction of multiple disulfide bridges (see 17). NAD-ME activity is also regulated, in this case allosterically, by a number of metabolites including adenylates (14). Yet modulation of these enzymes, apart from a crude on/off mechanism, could have no effect on photosynthetic flux in vivo. These observations are important in that they should engender a degree of caution when espousing the importance of a key enzyme in regulation of any metabolic pathway and raise the question as to why sophisticated mechanisms of enzyme regulation are necessary for such enzymes. One possible explanation lies in the evolutionary origins of C4 photosynthesis. There is not a single enzyme in the C4 pathway that does not have a homologue in C3 plants, leading to the hypothesis that the genes encoding these proteins have been recruited for the C4 process [see Introduction; (9, 33)]. The presence in C4 plants of these housekeeping enzymes at levels up to 100-fold higher than those P1: FUM March 30, 2001 302 18:0 Annual Reviews AR129-11 MATSUOKA ET AL found in C3 leaf cells, often still in their original C3 compartment, could play havoc with all manner of pathways of secondary metabolism (see 15). It is tempting to postulate that regulation of these nonlimiting enzymes preserves flux through minor pathways by conserving metabolites from the massive flood of carbon through C4 photosynthesis. Transgenic C4 Plants and the CO2 Pump A long-standing focus of interest in C4 photosynthesis has been the CO2 concentrating mechanism and the barrier to CO2 diffusion afforded by the bundle sheath/mesophyll interface (see 49). This aspect of the C4 mechanism is particularly pertinent to the introduction of C4 traits into C3 plants that lack the Kranz leaf anatomy common to terrestrial C4 plants. It has long been postulated that the relative levels of the enzymes of the CO2 pump in MCs and the enzymes of the Calvin cycle in BSCs, along with the permeability characteristics of BSCs, largely determine the efficiency with which C4 photosynthesis operates (49). Transgenic plants with altered PEPC/Rubisco ratios (45, 50) or altered bundle sheath inorganic carbon composition (35) provide the opportunity to test these hypotheses. If the content of Rubisco in a C4 leaf is progressively reduced without a commensurate effect on PEPC levels (as is the case in the transgenic Flaveria discussed above), one would intuitively expect the bundle sheath CO2 concentration to rise. This would result in an increase in leakage of CO2 from the bundle sheath and a decline in photosynthetic efficiency per net CO2 fixed. This is, in fact, what is observed when the Rubisco antisense Flaveria transgenics are analyzed using carbon isotope discrimination (50) or chlorophyll fluorescence and gas exchange (45). Carbon isotope discrimination allows the calculation of a leakiness parameter known as φ, which is the fraction of CO2 generated in C4 acid decarboxylation that subsequently leaks out of the bundle sheath (see 49). In transgenic Flaveria with a 40% reduction in Rubisco, this leakiness parameter increased by 50%. The increased ATP required to support this CO2 leakage was also evident in the quantum efficiency of the photosystems where plants with a 70% reduction in Rubisco content required approximately 28 quanta per CO2 fixed compared with 17 for wild-type leaves at saturating light in air (45). It has been postulated that an important requirement of the CO2 concentrating mechanism of C4 photosynthesis is the lack of equilibration of CO2 and bicarbonate in BSCs (29). This hypothesis is supported by the lack of appreciable carbonic anhydrase (CA) activity in BSCs of any C4 plant (6). Theoretically, by reducing the bicarbonate content of the bundle sheath inorganic carbon pool, the concentration of CO2 (the active species for fixation by Rubisco) is optimized and leakage of bicarbonate to MCs through plasmodesmata minimized (29). This hypothesis has been tested and the energetic ramifications assessed by ectopically expressing CA from tobacco in BSCs of Flaveria (35). Transgenic plants with two- to fivefold increases in CA activity in BSCs showed similar increases in leakiness to the Rubisco antisense transgenic plants discussed above and an approximately 20% P1: FUM March 30, 2001 18:0 Annual Reviews AR129-11 ENGINEERING OF C4 PHOTOSYNTHESIS 303 decrease in light-saturated photosynthesis (35). These experiments indicate the importance of seemingly minor aspects of biochemical specialization of the C4 pathway in determining the efficiency of the process. MOLECULAR ENGINEERING OF C4 ENZYMES IN C3 PLANTS How to Overproduce C4 Enzymes in the Leaves of C3 Plants Previously, attempts have been made to transfer C4 traits to C3 plants by conventional hybridization between C3 and C4 plants (4). However, this approach was available only in several plant genera such as Panicum, Moricandia, Brassica, Atriplex, and Flaveria. Moreover, most C3-C4 hybrids showed infertility due to abnormal chromosome pairing and/or genetic barriers. Recent developments in plant genetic engineering have enabled us to introduce the desired genes encoding C4 enzymes into C3 plants. In the past several years a variety of “C4 transgenic” C3 plants have been produced (Table 3). Enzymes Located in the Mesophyll Cells of C4 Plants The first attempt of this kind used a chimeric gene construct containing a cDNA of the maize C4-specific PEPC (Ppc cDNA) fused to the 50 - and 30 -flanking sequences of the chlorophyll a/b binding protein gene (Cab) from Nicotiana plumbaginifolia (25). The introduction of this chimeric gene into tobacco increased the PEPC activity in the leaves to 2.2-fold that of nontransformants, but the levels of transcripts and protein in these transformants were far below those in maize. Similarly, the expression of a cDNA for the C4 enzyme under the control of strong promoters such as Cab, rbcS, and Cauliflower mosaic virus 35S promoters led to only two- to fivefold increases in the activity of PEPC (20, 31), PPDK (12, 27, 44), and NADP-MDH (19). In these transformants, the level of the enzyme protein was low and only detectable by immunoblotting. The expression of bacterial Ppc genes from either Escherichia coli or Corynebacterium glutamicum under the control of the 35S promoter increased the enzyme activity of transgenic potato leaves but the extent of increase was less than several fold, a value almost comparable to that obtained with the higher plant Ppc cDNA under the control of the 35S promoter (20). To raise the expression level of the C4 enzymes, sequences that have enhancerlike effects were included in the introduced gene (20, 28). Gehlen et al (20) examined the effects of the 50 -untranslated region (UTR) of the chalcone synthase gene from parsley, and found that the expression of the Ppc gene from C. glutamicum under the control of the 35S promoter was enhanced by its presence. The highest expression level, however, was still only fivefold that of nontransformants in terms of PEPC activity. Thus, conventional strategies to express foreign genes in transgenic plants did not dramatically increase the activity of C4 enzymes in the leaves of C3 plants. P1: FUM March 30, 2001 304 18:0 Annual Reviews AR129-11 MATSUOKA ET AL TABLE 3 Increase in activities of C4 enzymes in the leaves of transgenic C3 plants C4 enzyme (Location in C4 plants) Host C3 plant PEPC (MC) Tobacco Potato Rice PPDK (MC) Tobacco Arabidopsis Potato Rice Highest activitya (Increase in fold) References Nicotiana Cab prom::maize C4 cDNA::Nicotiana Cab terminator 35S prom::maize C4 cDNA 35S prom::50 UTR::Corynebacterium gene Intact maize C4 gene 2.2 25 2.4 5.4 31 20 110 32 35S prom::Mesembryanthemum FL CAM cDNA Arabidopsis rbcS prom::maize C4 cDNA 35S prom::maize C4 cDNA Enhanced 35S prom::maize C4 cDNA Rice Cab prom::maize FL C4 cDNA Maize Pdk prom::maize FL C4 cDNA Intact maize C4 gene 1.6 44 2.4 27 4.0 5.4 27 28 5 13 5 13 40 13 Introduced construct NADP-MDH (MC) Tobacco 35S prom::Sorghum FL C4 cDNA 3 19 AspAT (MC) Tobacco 35S prom::C4 Panicum cDNA Intact C4 Panicum gene 3.1 43 20 —b Rice AspAT (BSC) Tobacco 35S prom::C4 Panicum cDNA 3.5 43 NADP-ME (BSC) Potato 35S prom::C3 Flaveria cDNA Rice Cab prom::rice FL C3 cDNA Rice Cab prom::maize FL C4 cDNA 7.1 34 5 48 30 48 70 46 0.5c 45a 0.5c 45a Rice PEP-CK (BSC) a Maize Ppc prom::Urochloa C4 cDNA Maize Pdk prom::Urochloa C4 cDNA Highest enzyme activities among the primary transgenic plants are listed. b c Rice M Nomura & M Matsuoka, unpublished observation. Highest activities of the secondary transgenic plants relative to the activity of Urochloa leaves are presented. MC, mesophyll cells; BSC, bundle sheath cells; prom, promoter; FL, full-length. P1: FUM March 30, 2001 18:0 Annual Reviews AR129-11 ENGINEERING OF C4 PHOTOSYNTHESIS 305 Another approach has been used to introduce the intact gene of C4 enzymes from C4 plants into C3 plants. Our previous studies have demonstrated that the promoters for maize C4-specific genes such as Ppc and Pdk can drive high-level expression of a reporter gene in transgenic rice plants in an organ-specific, MC-specific, and light-dependent manner as in maize (38–40). These results suggest that the rice plant possesses the regulatory factors necessary for high-level expression of C4-specific genes, and they imply that the introduction of the intact maize gene would lead to high-level expression of C4 enzymes in rice leaves. As expected, the introduction of the intact maize C4-specific Ppc gene containing all exons and introns and its own promoter and terminator sequences led to high-level expression of the PEPC protein in the leaves of transgenic rice plants (32). The majority (85%) of the transgenic rice plants showed PEPC activity in the leaves 2- to 30-fold that of nontransformants, whereas the remaining showed activity 30- to 110-fold that of nontransformants, or 1- to 3-fold that of maize leaves. The level of the PEPC protein accounted for 12% of total leaf soluble protein at most. In these transgenic rice plants, the levels of transcripts and protein and the PEPC activity in the leaves all correlated well with the copy number of the introduced gene. The levels of transcripts per copy of the maize C4-specific Ppc gene were also comparable in both maize and transgenic rice plants (32). These observations suggest that the maize C4-specific Ppc gene behaves in a qualitatively and also quantitatively similar way in both maize and transgenic rice plants. The introduction of the intact maize gene was also effective in expressing another C4 enzyme, PPDK, in rice leaves. The introduction of the intact maize C4-specific Pdk gene increased the PPDK activity in rice leaves up to 40-fold that of nontransformants or about half of the activity in maize (12). In a homozygous transgenic line, the PPDK protein accounted for 35% of total leaf soluble protein or 16% of total leaf nitrogen (12), much above the levels of foreign protein in transgenic C3 plants reported previously. Such high-level expression of the C4 enzymes in rice leaves could not be solely ascribed to the transcriptional activity of the maize gene, since the expression of a chimeric gene containing the full-length cDNA for the maize C4-specific PPDK under the control of either the promoter of the maize C4-specific Pdk or the rice Cab promoter increased the PPDK activity only up to several fold (12). It is suggested that one or more of the introns or the terminator sequence of the maize gene, or a combination of both, leads to high-level expression of the C4 enzyme by increasing the stability of the transcript (12). Maize and rice both belong to the Gramineae. Our recent study indicated that the introduction of the intact gene from other C4 gramineous plants could also lead to high-level expression of a C4 enzyme in transgenic rice plants. The activity of the cytosolic form of AspAT in rice leaves was increased to reach 20-fold that of nontransformants by introduction of the intact gene from Panicum miliaceum (M Nomura & M Matsuoka, unpublished observations). P. miliaceum is classified in the NAD-ME subtype of C4 plants, whereas maize is in the NADP-ME subtype. Thus, the intact genes from C4 gramineous plants, irrespective of the C4 subtype, will likely lead to high-level expression of the C4 enzymes in MCs of C3 gramineous plants. P1: FUM March 30, 2001 306 18:0 Annual Reviews AR129-11 MATSUOKA ET AL This strategy, however, has some limitation in that transgenes from phylogenetically closely related plants have to be used to achieve high-level expression of the C4 enzyme in C3 plants. The intact maize C4-specific Ppc gene was not expressed at high levels in tobacco leaves, because of incorrect transcription initiation (25). Not only incorrect initiation and termination of transcription but also incorrect splicing could occur when genes from monocots are introduced into dicots (see 21). Thus, phylogenetic distance may hamper the expression of genes from C4 plants in the leaves of C3 plants. Enzymes Located in the Bundle Sheath Cells of C4 Plants Unlike the C4 enzymes located in MCs of C4 plants, those located in BSCs can be expressed at high levels in MCs of C3 plants by the introduction of a chimeric gene containing the full-length cDNA for the C4 enzyme fused to the Cab promoter, which directs mesophyll-specific expression in C3 plants (42). The expression of the maize C4-specific NADP-ME cDNA under the control of the rice Cab promoter increased the activity of NADP-ME in rice leaves to 30- or 70-fold that of nontransformants (46, 48). The level of the NADP-ME protein was also increased to several percent of total leaf soluble protein (46, 48). Such high-level expression was unique to the cDNA for the C4-specific NADP-ME, and expression of the cDNA for the C3-specific isoform increased the activity only several fold (34, 48). The Flaveria C4-specific Me gene has a regulatory sequence in the 30 UTR that enhances its expression (37), and this sequence increases expression of a reporter gene when combined with heterologous promoters in the leaves of both C4 Flaveria and tobacco (S Ali & WC Taylor, unpublished information). The maize C4-specific Me gene may therefore contain such an enhancer element for high-level expression whereas the C3-specific gene does not. The expression of a cDNA of the C4-specific PEP-CK of Urochloa panicoides under the control of the maize C4-specific Ppc or Pdk promoter was also effective in increasing the activity of PEP-CK in MCs of rice leaves (45a). Recently, expression of the intact gene for C4 enzymes located in BSCs of C4 plants in C3 plants has also been addressed. When the intact gene for the mitochondrial AspAT of Panicum miliaceum, which is located in BSCs, was introduced into rice, high AspAT activity was detected in vascular tissues and BSCs of transgenic rice plants (M Nomura & M Matsuoka, unpublished observations). Similar results were observed with the PEP-CK gene from Zoysia japonica and β-glucuronidase activity under the control of the PEP-CK promoter, which was selectively detected in vascular tissues and BSCs (M Nomura & M Matsuoka, unpublished information). These results demonstrate that the C4-specific genes for the BSC enzymes can retain their property of cell-specific expression even in a C3 plant, rice, and they therefore suggest that C3 plants have a regulatory mechanism for gene expression of the BSC-specific C4 genes at their correct site. This fact is interesting in terms of evolutionary aspects of C4 photosynthesis, but it also indicates that the strategy to introduce intact C4-specific genes is not applicable to building the C4 pathway solely in MCs of C3 plants. P1: FUM March 30, 2001 18:0 Annual Reviews AR129-11 ENGINEERING OF C4 PHOTOSYNTHESIS 307 Physiological Impacts of Overproduction of C4 Enzymes in C3 Plants PEPC At present, there are four independent reports of transgenic C3 plants that overproduce PEPC in the MC cytosol (Table 3): two independent reports of transgenic tobacco plants expressing the maize C4-specific PEPC gene (25, 31), transgenic potato expressing a bacterial PEPC gene from C. glutamicum (20), and transgenic rice expressing the maize C4-specific PEPC gene (32). In the former three cases, PEPC activities in the leaves of 2- to 5-fold greater than wild-type levels were reported, whereas in the latter, activities up to 110-fold greater than wild-type were observed. In general, PEPC from either higher plants or bacteria undergoes regulation by various metabolite effectors, being inhibited by malate, aspartate, and glutamate (see 17). Since concentrations of these inhibitors are high in the MC cytoplasm of C3 plants, about 1 mM for malate and around 40 mM for aspartate and glutamate (24), the PEPC activities of the transformants in vivo would be lower than maximum extractable activities, especially when measured in the presence of activators. Plant PEPC is also regulated by phosphorylation at a specific serine residue near the N terminus that reduces sensitivity to these inhibitors (see 17). The maize PEPC in transgenic rice leaves remained in its dephosphorylated and less active form during illumination (13). All the transformants analyzed to date show a higher level of malate (23, 25, 31) or OAA (13) in the leaves compared with wild-types, an indication that the foreign PEPC is at least active in the leaves of these transformants. The level of malate/OAA in these transformants, however, did not exceed twofold that of wildtype, irrespective of the expression level of PEPC in the C3 leaves. This is consistent with the notion that PEPC is not fully active in these plants. It is also likely that malate and OAA produced by the action of PEPC are metabolized inside the cell or translocated from the leaves. The endogenous PEPC in MCs of C3 plants has an anaplerotic function that replenishes the tricarboxylic acid (TCA) cycle with organic acids to meet the demands of carbon skeletons for amino acid synthesis (7). Thus, levels of organic acids other than malate and oxaloacetic acid, as well as foliar amino acids, would be predicted to increase in the transformants. At present, only a slight increase in the level of amino acids has been reported in transgenic potato (23). Effects of overexpression of PEPC on photosynthesis are controversial. At temperatures optimal for plant growth, practically no difference in the rate of CO2 assimilation and the CO2 compensation point (0) was observed in transgenic tobacco expressing the maize PEPC gene (25, 31). Activities of PEPC were only about twofold higher than in wild-type plants in these experiments. In transgenic rice plants expressing the maize PEPC gene, the rate of CO2 assimilation was also not altered significantly, but the O2 inhibition of net CO2 assimilation was mitigated with increasing activity of PEPC (32). However, this effect appears not to have resulted from the fixation of CO2 for photosynthesis by the maize PEPC. The initial CO2 fixation product, determined by 14CO2 labeling experiments with P1: FUM March 30, 2001 308 18:0 Annual Reviews AR129-11 MATSUOKA ET AL transgenic rice plants having a 50-fold elevation in PEPC activity, was exclusively the C3 compound 3-phosphoglycerate (13). In addition, overexpression of C4 PEPC in transgenic rice leaves suppressed CO2 assimilation at 2% O2 to a greater extent than that at 21% O2, probably through limitation of inorganic phosphate (Pi) for the Calvin cycle reactions (13). The major increase in PEPC activity may lead to depletion of Pi in the cytosol, through a stimulation of glycolysis that would suppresses sucrose synthesis. These collective reactions consume one Pi molecule and release four Pi molecules, respectively. Changes in the photosynthetic characteristics at optimal temperatures have been reported only in transgenic potato expressing the bacterial PEPC (23). In this case, it was reported that the CO2 compensation point independent of respiration (0 ∗ ), measured according to Brooks & Farquhar (3), decreased by about 16% in the transformants with fivefold activity, as compared with wild-type plants (23). The authors argue that the decrease in 0 ∗ resulted from the increase in CO2 concentration in the vicinity of Rubisco by decarboxylation of organic acids produced by PEPC and/or through its anaplerotic function (23). Determination of the initial CO2 fixation products and quantification of flux through PEPC in these potato transformants is awaited. Significant changes in the photosynthetic characteristics in PEPC transformants have thus far been observed mostly at supraoptimal temperatures. In wild-type plants, the rate of CO2 assimilation in air declines with increasing temperature, whereas in PEPC-overexpressing transformants, it remains unchanged or becomes even greater at higher temperatures in transgenic tobacco (31) and potato (34). This phenomenon is not yet fully understood, but it might be possible that PEPC participates in the initial CO2 fixation for photosynthesis (despite the absence of elevated levels of the other enzymes necessary for a C4 cycle) or that it acts to increase the CO2 concentration in the vicinity of Rubisco under conditions in which the oxygenation reaction of Rubisco proceeds much faster that its carboxylation reaction, as proposed previously (23, 34). PPDK There are four reports of transgenic C3 plants that express PPDK in the MC chloroplast (Table 3): transgenic Arabidopsis (27), potato (28), rice (12) expressing the maize C4-specific PPDK gene, and transgenic tobacco expressing a PPDK gene from the CAM plant Mesembryanthemum crystallinum (44). In all cases, no changes in photosynthetic characteristics were observed in these transformants, even in the transgenic rice with PPDK activity 40-fold higher than wildtype levels (12). A modest increase in the δ 13C value was reported in the transgenic potato but this difference was marginal in significance (28). Some changes in the level and composition of free amino acids were also reported in the transgenic tobacco (44). In general, the overall PPDK reaction is freely reversible, depending on concentrations of substrates, activators, and inactivators (5). This is probably the case in MCs of C3 plants, in which the activity of inorganic pyrophosphatase and adenylate kinase is low (22) and could be the reason why the overexpression of PPDK does not result in significant effects on carbon metabolism in C3 leaves. P1: FUM March 30, 2001 18:0 Annual Reviews AR129-11 ENGINEERING OF C4 PHOTOSYNTHESIS 309 The expression of chloroplast-targeted PPDK increased the number of seeds per seed capsule and the weight of each seed capsule by about 40% and 20%, respectively, in transgenic tobacco, with about a 1.5-fold increase in activity of PPDK in the leaves (44). These effects were not observed in transgenic tobacco plants that express PPDK in the cytosol (44). The mechanism of the increase in seed yield by PPDK is obscure at present. One possibility is that overexpression of PPDK in the chloroplast enhances photosynthesis in organs surrounding seeds. In seed pods of C3 dicots and spikelets of C3 monocots, enzyme activities associated with the C4 pathway are high and C4-like photosynthesis is operative, contributing significantly to grain filling (see 26, 30). Thus, it is possible that overexpression of PPDK in the chloroplast enhances C4-like photosynthesis in organs such as hulls and ears to raise the yield of seeds and grains. NADP-ME There are four reports of transgenic C3 plants that express NADP-ME in the MC chloroplast (Table 3): two sets of transgenic rice plants expressing the maize C4-specific isoform (46, 48), transgenic rice expressing the rice C3-specific isoform (48), and transgenic potato expressing the C3-specfic isoform of Flaveria pringlei (34). The transformants expressing the C3-specific isoform with activities up to several fold higher than wild-type levels did not show any detectable differences in their growth and photosynthesis (34, 48), whereas those overexpressing the maize C4-specific isoform showed serious stunting and leaf photobleaching, due to increased photoinhibition of photosynthesis under natural light conditions (46, 48). It is proposed that the maize C4 NADP-ME in the chloroplasts acts to increase the NADPH/NADP ratio and to suppress photorespiration, rendering photosynthesis more susceptible to photoinhibition (46, 48). Such detrimental effects of the maize enzyme might imply significant flexibility of carbon metabolism in MCs of C3 plants, especially in terms of transport of metabolites between the cytosol and the chloroplast stroma. PEP-CK There is only one report of transgenic rice plants that express the C4specific PEP-CK of U. panicoides in the MC chloroplast (45a) (Table 3). Although this enzyme is located in the BSC cytosol of U. panicoides, the introduced construct was designed so that the enzyme was targeted to the MC chloroplasts in transgenic rice leaves. The expression of chloroplast-targeted PEP-CK showed significant alterations of carbon metabolism in rice leaves (45a). In 14CO2 pulsechase experiments of transgenic rice with PEP-CK activity comparable to that in U. panicoides, about 20% of the radioactivity was incorporated in the C4 compounds, malate, OAA, and aspartate. Feeding of 14C-labeled malate also increased the incorporation of the radioactivity into sucrose. It is unclear whether the introduced PEP-CK together with endogenous PEPC could drive a C4-like pathway in MCs of rice leaves as proposed previously, since the CO2 compensation point was unchanged in transgenic rice plants (45a). P1: FUM March 30, 2001 310 18:0 Annual Reviews AR129-11 MATSUOKA ET AL FUTURE PERSPECTIVES There has been considerable progress in recent years in the molecular engineering of C4 photosynthesis. The technology to express the C4 enzymes at high levels and in the desired locations in the leaves of C3 species is becoming well established, and it is now possible to produce transgenic C3 plants that express at least a set of key enzymes of the C4 pathway. Thus, we have just reached the starting point in introducing the basic biochemical elements of the C4 pathway into C3 plants. Apart from the goal of installation of a complete C4 pathway into C3 plants, some transgenic C3 plants that overproduce a single C4 enzyme show alterations in carbon metabolism. These plants are also proving to be useful tools in probing the “housekeeping” function(s) of the C4-like enzymes in C3 plants and the evolution of the C4 photosynthetic genes. Experiments with transgenic plants have reinforced the fact that the C4 mechanism is a finely tuned metabolic “machine” where both a high degree of precision in gene expression and structural morphology work together to concentrate CO2 efficiently at the site of Rubisco. Work with transgenic C4 Flaveria and also transgenic C3 plants shows that relatively small changes in leaf biochemistry, induced by transgene action, can have major deleterious effects on photosynthetic competence. In light of these observations, some important questions must be answered in the quest to introduce the C4 pathway in C3 crop plants. First, can we deliver the degree of precision required to coexpress the necessary genes at the correct levels and ratios in the correct compartments? For the primary enzymes of the C4 pathway these preliminary results are promising but correct posttranslational regulation of the introduced, heterologous enzymes, fine-tuning of the levels of ancillary enzymes (such as CA, adenylate kinase, and pyrophosphatase) and metabolite transporters must also be addressed. Most important, can we create an efficient CO2 concentrating mechanism in a plant lacking Kranz leaf anatomy, a morphological feature independently arrived at several times through the convergent evolution of C4 plants? Would the metabolic cost of establishing a CO2 concentrating mechanism without an effective barrier to CO2 diffusion outweigh the advantages? In connection with this key issue, there are good examples of higher plant CO2 concentrating mechanisms without Kranz anatomy, namely, the submersed aquatic macrophytes (SAMs) such as Hydrilla verticillata, in which an intracellular C4-like pathway is induced in response to a decline of ambient CO2 concentration (see 36). It remains to be seen, however, whether this process is a highly efficient addition to the C3 process or a lowefficiency survival mechanism in SAM species. Studies on the mechanisms of induction of a C4-like pathway in SAM plants may help us to understand how to introduce an effective C4-like mechanism to MCs of C3 plants. However, a conclusive answer as to the performance of an artificially introduced C4 pathway in C3 crops can only be obtained by the generation and comprehensive analysis of transgenic crop plants such as those described here and those currently being produced. 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