Enhancing Photosynthesis with Sugar Signals

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Opinion TRENDS in Plant Science Vol.6 No.5 May 2001 197

to sophisticated fine control mechanisms represented Enhancing the best targets because they exerted the most control on flux. In the Calvin cycle, through which carbon enters the reactions of photosynthesis and photosynthesis with metabolism, there are four key irreversible enzymes: Rubisco, in the carboxylation phase of the cycle, and fructose-1,6-bisphosphatase (FBPase), sugar signals sedoheptulose-1,7-bisphosphatase (SBPase) and phosphoribulokinase (PRK), in the regenerative phase (Fig. 1). In experiments on transgenic tobacco and Matthew Paul, Till Pellny and Oscar Goddijn potato expressing antisense cDNA to each of these enzymes, a decrease of 50% maximum catalytic enzyme activity had minimal impact on the rate of Photosynthesis has long been a target in the quest to maximize crop photosynthesis under standard growth-room productivity to feed burgeoning populations. Recent evidence suggests that conditions1–4. The most extreme of these is PRK, 85% improved photosynthetic performance can be most easily achieved by of whose maximum catalytic activity can be removed modifying sugar-signalling mechanisms that control the expression of genes before there is any effect on photosynthesis4. This is for whole pathways and processes that determine photosynthetic capacity and because sophisticated fine control of PRK can source–sink balance, rather than by directly targeting individual ‘key’ enzymes. overcome a large genetic decrease in PRK protein4,5. Here, we highlight recent progress and support for the hypothesis that genetic The full story of control of Calvin cycle flux comes from modification of trehalose metabolism through its interaction with sugar- analysis under a range of conditions that plants are signalling pathways can enhance photosynthetic capacity. likely to encounter in the natural environment6,7. This emphasizes that control exerted on irreversible Calvin For a long time, one of the aims of plant scientists has cycle enzymes occurs through allosteric and post- been to improve photosynthesis to maximize crop translational control mechanisms, and that targeting productivity. The logic of the argument is clear: the these enzymes for genetic modification is problematic yield of crops broadly relates to the amount of light because of the counteracting fine control mechanisms. intercepted and the cumulative photosynthesis over a Another way in which control of enzyme activity is growing season. If photosynthesis could be increased exerted is through expression level. The catalytic then it would be possible to capitalize on and extend activity of reversible enzymes not subject to fine this relationship. Breeding and agronomic practice control is regulated in this way, and research using have effectively achieved this over the past century by transgenic plants has shown that reversible enzymes creating the conditions under which photosynthesis have the potential to exert a high degree of control. can flourish. New varieties have been bred with bigger This is true for aldolase, for example7. However, it is assimilate sinks and with better coordination of leaf not just the catalytic activity of reversible enzymes production with the amount of light available during that is controlled by gene regulation. A second the growing season. High fertilizer use has further important conclusion of plant science over the 1990s, facilitated a high photosynthetic rate and, in the which developed from several approaches and has horticulture industry, ‘fertilization’of tomato crops been confirmed by work on the response of plants to

with CO2 is used routinely to maximize carbon fixation. elevated CO2, is that the control of photosynthesis at As our knowledge of plant biochemistry, the molecular level by sugar and nitrogen signals metabolism and, latterly, molecular biology has through changes in whole plant carbon–nitrogen increased, our sights have shifted towards more balance8–10 overrides the control of photosynthesis by targeted manipulation of metabolism itself. If we other mechanisms. This gives us clues to how to could find out which are the ‘key’ enzymes responsible proceed with the genetic modification of for controlling photosynthetic rate or those that lead photosynthesis. Direct genetic manipulation of

to photorespiratory losses of the CO2, it would be photosynthesis by targeting key enzymes, even if it possible to target these enzymes to improve were possible to overcome post-translational control

Matthew Paul* photosynthesis. Isolation of the genes and genetic mechanisms and the flexibility of metabolism and to Till Pellny transformation would enable photosynthetic carbon increase the photosynthetic rate, could be frustrated Crop Performance and acquisition to be increased directly. The reasoning by the overproduction of a sugar signal that repressed Improvement, IACR- behind this was good, as far as we knew at that time. expression of photosynthetic genes. To achieve Rothamsted, Harpenden, Hertfordshire, UK AL5 2JQ. significant positive changes in photosynthesis and *e-mail: The last will be first and the first last in metabolic resource allocation, it will be necessary to understand [email protected] regulation the signals and molecular mechanisms that control Oscar Goddijn Genetic modification of plant metabolism proceeded the expression not just of aldolase, PRK or Rubisco, Syngenta Mogen, apace during the 1990s and several strong messages but of whole pathways and suites of genes that Einsteinweg 97, 2333 CB have come from this. One is that our concept of ‘key’ determine photosynthetic capacity. These Leiden, PO Box 628, 2300 AP Leiden, The enzymes has changed drastically. It was thought that mechanisms underpin source–sink interactions and Netherlands. enzymes that were essentially irreversible and subject productivity in plants.

homologies to microbial TPS genes can be found in

Ru5P PRK CO2 the Arabidopsis genome, one of which has been cloned and shown to synthesize T6P (Ref. 12). This Isomerase 5 Ru1,5BP raises the question of what the function of these 5 R5P 5 Epimerase Rubisco genes and of trehalose metabolism in plants are. Xu5P In yeast, trehalose metabolism performs a signalling role13, although the precise mechanistic Transketolase 3PGA 3 5 details of this are still being unravelled, as are Glycerate-3-P kinase interspecific differences between yeast species and other microorganisms14. Evidence from our current S7P 7 Calvin cycle research suggests that a similar role has persisted 3 1,3BPGA 15 SBPase in plants . In experiments in which E. coli otsA and otsB genes, which encode TPS and TPP, Glyceraldehyde-phosphate S1,7BP 7 dehydrogenase respectively, were expressed in transgenic tobacco (Nicotiana tabacum), there were effects consistent E4P with an effect on sugar signalling. In plants Aldolase GAP 3 expressing E. coli TPS, rates of photosynthesis per 4 Transketolase Triose phosphate unit leaf area are increased by up to a third under isomerase saturating irradiance; in plants expressing TPP, Aldolase 15 F6P 6 photosynthesis is decreased by a similar amount . This shows that it is possible to increase the FBPase F1,6BP 6 DHAP Triose phosphate photosynthetic capacity of plants. Measurements are exported 3 continuing to determine the biochemical and

TRENDS in Plant Science molecular basis of these results but the data are consistent with the hypothesis that genetic Fig. 1. The Calvin cycle of plants, the entry point of carbon in photosynthesis and metabolism and modification of trehalose metabolism has affected initial entry point of carbon into the food chain. The cycle is composed of 13 reactions catalysed by photosynthetic capacity through sugar signalling. 11 enzymes, and consists of three distinct phases. The first phase is carboxylation (blue arrows), catalysed by Rubisco. The second phase is the reductive part (red arrows), during which ATP and Our model to explain the findings centres on T6P. NADPH derived from the light reactions are used to convert 3-phosphoglyceric acid (3PGA) into In yeast, T6P has a role in controlling glycolysis and glyceraldehyde phosphate (GAP). The third phase (black arrows) regenerates ribulose-1,5- sugar signalling through its interaction with bisphosphate (Ru1,5BP) for the carboxylation reaction. Enzymes that are essentially irreversible hexokinase, a putative sensor in yeast and plants13. and that are traditionally classified as ‘key’ enzymes are highlighted in red [Rubisco, fructose-1,6- bisphosphatase (FBPase), sedoheptulose-1,7-bisphosphatase (SBPase) and phosphoribulokinase T6P has not been previously found in plant tissue, (PRK)]. Reversible enzymes are highlighted in blue. Abbreviations: 1,3BPGA, although this might be because researchers have not 1,3-bisphosphoglyceric acid; 3PGA, 3-phosphoglyceric acid; DHAP, dihydroxyacetone phosphate; been looking and because methods have not been E4P, erythrose-4-phosphate; F1,6BP, fructose-1,6-bisphosphate; F6P, fructose-6-phosphate; GAP, sensitive enough to detect it. We have developed a glyceraldehyde phosphate; R5P, ribose-5-phosphate; Ru1,5BP, ribulose-1,5-bisphosphate; Ru5P, ribulose-5-phosphate; S1,7BP, sedoheptulose-1,7-bisphosphate; S7P, sedoheptulose-7-phosphate; sensitive assay for T6P in plants, using a hexokinase Xu5P, xylulose-5-phosphate. Numbers highlighted in grey represent the number of carbon atoms in isoform from Yarrowia lipolytica. The hexokinase II each molecule. from this yeast is particularly sensitive to inhibition by T6P and forms the basis of an accurate method to Signals from trehalose metabolism quantify T6P (Ref. 16). Our data using this assay In addition to the change in thinking about the technique have also been confirmed by HPLC control of metabolic fluxes that we did know about, a measurements. In TPS lines with enhanced rates of further surprise of plant metabolism over the past photosynthesis, the T6P content is fivefold higher. two or three years has been the finding that the This is approaching this range of other genes for trehalose metabolism are universal in phosphorylated intermediates in plants. plants11. Trehalose (α-D-glucopyranosyl α-D- In this model, T6P interacts with plant glucopyranoside), a non-reducing disaccharide hexokinase as it does in yeast13 (Fig. 2). Plants have consisting of two molecules of glucose, is the major several isoforms of hexokinase and T6P might disaccharide of microorganisms, fungi and insects, interact with just one of these. We suggest that T6P but has been largely superseded by sucrose in plants interacts with hexokinase, modifying perception of (although trehalose does accumulate in some glucose content. In plants expressing E. coli TPS and resurrection species). Trehalose is synthesized from hence with higher T6P content, a carbon deficit is glucose-6-phosphate (G6P) and uridine-5- perceived through interaction with hexokinase and diphosphoglucose (UDPG) in a reaction catalysed by photosynthetic capacity is enhanced. Plants trehalose phosphate synthase (TPS) (Fig. 2). The expressing E.coli TPP, by contrast, have levels of T6P phosphate group is removed from the trehalose-6- that are below detection. Low T6P content might phosphate (T6P) formed in this reaction sequence by mean that these plants sense a surfeit of glucose and trehalose phosphate phosphatase (TPP). It is now hence that the expression of genes for photosynthesis clear that the genes are widespread if not universal is decreased. Clearly, more work is required to in higher plants. At least five distinct sequences with substantiate this hypothesis. In fact, the functioning

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the transgene is free to interact with other enzymes Photosynthesis and proteins within the cell. Thus, cells are exposed to a signal that is normally within the enzyme Calvin Starch Resource complex. ‘Normally’ might be the key word here: cycle allocation plant TPS, like SPS, interacts with 14-3-3 proteins20. Triose These are thought to be involved in the coordination Hexose Chloroplast phosphate of metabolism in response to carbon supply21 and affect protein–protein interactions. Loss of 14-3-3 14-3-3 Proteins Sugar binding under conditions of low carbon supply21 Trehalose-6- sensing might affect the integrity of SPS–SPP and TPS–TPP TPP phosphate TPS cycle protein complexes. Dissociation of the complexes under carbon starvation would liberate T6P to Trehalose UDPG + G6P SnRK1 interact with enzymes and proteins such as Trehalase hexokinase. A similar scenario for S6P can be Energy envisaged. The effects of S6P on metabolism as for Hexose HXK Hexose-P T6P in plants are largely unknown. When interpreting the results, it is also important UDPG + F6P Glycolysis to bear in mind that any effect on hexokinase will have an effect on metabolite flux and metabolite Sucrose Amino content downstream of hexokinase, which might in acids turn affect sugar signalling. G6P, for example, the Invertase Export Sinks product of hexokinase, does not exert feedback control on hexokinase in plants and fungi as it does in TRENDS in Plant Science animals, but it does affect the activity of SnRK1 Fig. 2. Interaction of trehalose-6-phosphate (T6P) with hexokinase (HXK) and sugar signalling (SNF1-related protein kinase1)22, a plant protein pathways. T6P is synthesized from uridine-5-diphosphoglucose (UDPG) and G6P, kinase related to SNF1 (sucrose nonfermenting1) catalysed by trehalose phosphate synthase (TPS). The phosphate group is removed from T6P by from yeast. SnRK1 has been implicated in changes trehalose phosphate phosphatase (TPP). The reaction sequence is similar to that of sucrose, except that fructose-6-phosphate (F6P) substitutes for G6P in sucrose synthesis. HXK, sucrose- in the activities and expression of enzymes involved nonfermenting-1-related protein kinase1 (SnRK1) and 14-3-3 proteins are components of a sugar- in sucrose and starch metabolism in response to sensing complex in plants. This putative complex underpins source–sink interactions and enables carbon supply23. plants to redistribute resources between processes that produce carbon compounds in photosynthesis and those that store, mobilize and consume carbon in response to changing The plant material offers an unprecedented source–sink relationships caused by environment and development. Broken arrows indicate the link opportunity to study the interactions between between HXK, sugar sensing and photosynthesis; dotted green line indicates putative trehalose- metabolism, sugar signalling and resource allocation synthesizing complex. at the molecular, biochemical and physiological levels. In particular, it might be possible to isolate of plant hexokinases and their role in sugar sensing transcription factors that coordinate expression of requires more detailed characterization. What is the many genes that determine photosynthetic clear, however, is that the function of hexokinase in capacity and leaf development. Indeed, isolation plants is subtly different to that in heterotrophs. of transcription factors might be a more readily Plants can obtain hexose phosphate directly from attainable goal than understanding the complex photosynthesis without the need for hexokinase. mechanistic details of how signals arising from Plant hexokinase functions in the re-entry of hexose trehalose metabolism can enhance photosynthetic into the hexose phosphate pool, mainly from sucrose rate. and starch breakdown. Hexokinase might be part of cycles of sucrose and starch synthesis and Conclusions breakdown that are part of the mechanism by which We have shown that it is possible to enhance plants sense carbohydrate status and adjust resource photosynthetic capacity and photosynthetic rate in 10 Acknowledgements allocation accordingly . transgenic plants through the insertion of the IACR receives grant-aided There are strong parallels between trehalose E. coli TPS gene otsA. There are many questions support from the and sucrose metabolism that might help in still to be resolved, but the main purpose of this Biotechnology and interpreting the findings. Sucrose phosphate article is to highlight the potential significance of Biological Sciences Research Council of the synthase (SPS) and sucrose phosphate phosphatase the research. Evidence is accumulating that an UK. We are indebted to (SPP) exist as a complex17. This might ensure ancient and once obscure metabolic pathway in Klaus-Peter Krause for his efficient substrate channelling and that, under most plants has a role in sugar signalling. Full part in setting up the collaboration between the conditions, sucrose-6-phosphate (S6P) is only seen by understanding of the mechanistic basis of enhanced laboratories. Our SPP. A similar enzyme complex has been shown for photosynthesis in these plants will come from a gratitude extends to trehalose synthesis in yeast and again, under most detailed analysis of the interaction of metabolism Carlos Gancedo (Madrid, conditions, T6P remains within the complex18. In with sugar signalling pathways. Of particular Spain) for providing us 19 with Yarrowia lipolytica E. coli, TPS and TPP do not form a complex and, in interest will be the characterization of plant hexokinase. plants expressing E. coli TPS, T6P synthesized by hexokinase isoforms, plant TPS and TPP, and how

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they interact with each other and with metabolism, sugar signals and molecular mechanisms that and sugar signalling and its effect on resource underpin source–sink interactions and crop allocation in plants and crops. productivity. An opportunity now presents itself to It is particularly exciting that the transgenic plant enhance the photosynthesis of crops by manipulating material gives us the opportunity to learn more about sugar signals and signalling pathways.

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Most closely related to ciliates and dinoflagellates1,2, The apicoplast: a new the phylum Apicomplexa constitutes a particularly diverse and ancient group of parasitic protists. They are classified as protozoa (>4000 species) that mostly member of the have an obligately intracellular lifestyle. Some are important causative agents of human and animal diseases, the most potent of which is Plasmodium, plastid family the agent of malaria, recognized by the World Health Organization as being in the top three killers in the world. Toxoplasma gondii and Cryptosporidium are Eric Maréchal and Marie-France Cesbron-Delauw major opportunistic pathogens in immunocompromised patients. Others cause economically important animal diseases such as Protozoan parasites of the phylum Apicomplexa include pathogens such as babesiosis (Texas water fever), coccidiosis and Plasmodium, Toxoplasma and Cryptosporidium. They have been shown to theileriosis (East Coast fever). In spite of sustained contain a vestigial nonphotosynthetic plastid, the apicoplast, which might efforts, no efficient vaccine is available and have arisen by secondary endosymbiosis. Little is known about the function of chemotherapy is facing the emergence of drug the apicoplast but the parasites exhibit delayed cell death when their resistance, which is becoming crucial in malaria apicoplast is impaired. The discovery of the apicoplast opens an unexpected treatment. opportunity to link current fundamental research on plant and algal plastids to Apicomplexan parasites have complex life the physiology of apicomplexans. For example, the apicoplast might provide cycles that involve both asexual and sexual new targets for innovative drugs that act as herbicides and do not affect the reproductions, with many variations in the mammalian host. different groups. Apicomplexans are transmitted