Photorespiration

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PHOTORESPIRATION: A WASTEFUL R ELIC, OR A POTENTIALLY USEFUL METABOLIC TOOL? BY LISA KEENAN A Thesis Submitted to the Division of Natural Sciences New College of Florida In partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Prof. Amy Clor e, Ph.D, Associate Professor of Biology Sarasota, Florida May 2010

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PHOTORESPIRATION: A WASTEFUL R ELIC, OR A POTENTIALLY USEFUL METABOLIC TOOL? Lisa Keenan New College of Florida, 2010 ABSTRACT This thesis is an in-depth review of the photorespiratory (C2) metabolic pathway, as well as the other pathways closely connected to it, and the recent advances in the quest to produce a more productive plant. There has been some contention in the scientific community about the usefulness of the C2 pathway: on the one hand, it is a pathway that drains energy while producing no G3P (g lyceraldehyde 3-phosphate), a molecule necessary for the production of sugars, fatty acids, and amino acids; on the other hand, significant research has connect ed the photorespiratory meta bolism with stress protection, nitrogen metabolism, and the equilibrium of reactive oxygen species (ROS) generated in the plant cell, which has been found to im pact signaling pathways. In the field of engineering productive plants, a significant quantity of research has focused on reducing or eliminating the photorespirative pathway with small success to date. However, a new tactic of increasing the metabolic rate of multiple pathways, the C2 included, shows promise. Dr. Amy Clore Division of Natural Sciences ___________________________________

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Acknowledgements I would like to thank my family, Anne, John, Lauren and Rebecca Keenan for all their encouragement and moral support. Thank you all for cheering me on and supporting me while I was working; I couldnt have done it without all of you. I have tremendous gratitude for the guidance of Professor Amy Clore, my advisor and thesis sponsor. You recognized and assisted me through my rough patches, and were highly knowledgeable and supportive. A student couldnt ask for a better advisor. Id also like to thank Professor Paul Scudder, faculty sponsor and previous advisor. There were a few times where things felt bleak, yet you kept me in line and helped me see the light at the end of the tunnel. To Professor Beulig, who has been exceptionally understanding througho ut this process. I greatly appreciate your ki ndness and consideration. ii

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Table of Contents List of Illustrations v List of Equations v 1. Photosynthesisan overview 1 1.1 Introduction 1 1.2 Photosynthesisan overview 3 1.3 The light reactions 6 1.4 The light independent reactions 11 1.4.1 Carbon fixation 13 1.4.2 Reduction reactions 13 1.4.3 Regeneration 14 1.5 Rubisco 15 1.5.1 Rubisco structure 19 1.5.2 Rubisco reaction mechanisms 22 1.6: Photorespirationan overview 24 1.7 Alternate strategies for photosynthesis 28 1.7.1 C4 metabolism 28 1.7.2 CAM photosynthesis 30 1.8 Conclusions 31 2. The C2 pathway and relevant mutants 32 2.1 Introduction 32 2.2 The effects of suppressing photorespiration 32 2.3 Photorespiratory mutants 33 2.3.1 Carbon recycling photorespiratory enzymes 34 2.3.1.1 Phosphoglycolate phosphatase 34 2.3.1.2 Glycolate oxidase 38 2.3.1.3 Glutamateand serine-g lyoxylate aminotransferase 42 2.3.1.4 Glycine decarboxylase and serine hydroxymethyltransferase 45 2.3.1.5 Hydroxypyruvate reductase 49 2.3.1.6 Glycerate kinase 51 2.3.2 Nitrogen uptake/reuptake 52 2.3.2.1 Glutamine synthetase 54 2.3.2.2 Glutamate synthase 58 2.3.3 H2O2 metabolism 60 2.3.3.1 Catalase 60 2.4 Conclusions 64 3. Efforts to improve photosynthetic efficiency 65 3.1 Introduction 65 3.2 Efforts to reduce the C2 pathway 67 3.2.1 Rubisco 67 3.2.1.1 Site-directed mutagenesis 68 3.2.1.2 The concept of directed protein evolution .71 3.2.1.3 Horizontal transfer 72 iii

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3.2.2 Efforts to im prove Rubisco activase 74 3.2.3 Efforts to convert C3 plants into C4 plants 76 3.3 Efforts to increase metabolic rate 81 3.3.1 The Calvin cycle 81 3.3.2 The photosystems 88 3.3.3 The photorespiratory pathway 91 3.4 Conclusions 94 4. Reactive oxygen species 96 4.1 Introduction 96 4.2 ROS production in the plant cell 96 4.2.1 The chloroplast 97 4.2.2 The peroxisome 100 4.2.3 The mitochondrion 100 4.2.4 The apoplast 101 4.2.5 The cytosol 101 4.3 Photorespiration and ROS 101 4.4 ROS signaling of the MAPK kinase pathway 103 4.5 Conclusions 106 5. Possible future initiatives 108 5.1 Introduction 108 5.2 Metabolic mutants 108 5.3 Future studies 110 5.4 Conclusions 112 Appendix: Abbreviations .113 Works Cited .117 iv

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v List of Figures Figure 1.1 A basic schematic of the C2 and C3 pathways .2 Figure 1.2 The basic structure of a chloroplast 4 Figure 1.3 The light reactions 6 Figure 1.4 A brief overview of the Calvin cycle 12 Figure 1.5 The four forms of Rubisco 17 Figure 1.6 The Rubisco active site 22 Figure 1.7 The RuBP mechanisms 24 Figure 1.8 The photorespirative pathway 26 Figure 1.9 The C4 Kranz anatomy .29 Figure 2.1 The 2PG metabolism in Synechocystis 37 Figure 2.2 The GS-GOGAT cycle 53 Figure 3.1 A diagram of systems modified in the effort to improve efficiency 66 Figure 3.2 The plant and bacterial C2 pathways 92 Figure 4.1 A diagram of the e volution of ROS in different cellular structures 97 Figure 4.2 The Mehler reaction, or water-water cycle 98 Figure 4.3 A generic depiction of the MAPK signal transduction pathway List of Equations Equation 1.1 The overall r eaction of photosynthesis ............ 3 Equation 1.2 The Rubisco oxygenic reaction 3 Equation 1.3 Summary of the light reactions 5 Equation 1.4 The light-independent reactions 5 Equation 1.5 The ratio of Rubi sco carboxylation to oxygenation 25 Equation 2.1 The glycine decarboxylase reaction 46 Equation 2.2 The overall catalase reaction 60 Equation 2.3 The catalase reaction, stage 1 61 Equation 2.4 The catalase reaction, stage 2 61 Equation 4.1 The Mehler reaction 99 Equation 4.2 The superoxide dismutase reaction 99 Equation 4.3 The ascorbate peroxidase reaction 99

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1 Chapter 1. Photosynthesisan overview 1.1 Introduction The kingdom Plantae consists of auto trophic, multicellular organisms that produce food through photosynthesis. Plants are vital to the survival of all heterotrophs due to the fact that their great numbers serv e as part of the base of the trophic pyramid. Humans directly re ly on plants for oxygen, food, medicines, and clothing to list just a few of their us es. It is vital to unde rstand how these life forms survive and function due to the many great roles plants play in the environment and human well-being. These dynamic organisms’ ability to largely sustain an entire ecosystem demands a deep understanding the many components and pathways that make their existenc e possible. The special processes and constituents of the average successful plan t are the product of b illions of years of trial and error, time and evolution. This thesis will focus mainly on photorespiration, a seemingly wasteful proces s that may play important roles that were, until recently, underappreciated. This first chapter will go into detail explaining some of the most important pathways in plant photosynthesis, incl uding the light indepe ndent reactions, the C3 or Calvin Cycle with special focus on the enzyme Ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco), the C4 cycle, and crassulacean acid metabolism (CAM). A list of related abbrevia tions is provided in the appendix.

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2 Figure 1.1: A basic schematic of the C2 and C3 pathways. Notice the Calvin cycle’s net gain of carbon, and the photor espiratory pathway’s net loss of carbon (Taiz and Zeiger,1998) The focus of the remainder of th is thesis, photorespiration, is the biochemical pathway resulting from the oxygenation of ribulose 1,5-bisphosphate (RuBP) by Rubisco. It has been the sour ce of some controversy, as will be addressed. This biochemical pathway has been viewed in both a negative and positive light by the scientific community, with substantial data and arguments evidencing both perspectives. On the one hand, the pathway can be viewed as simply a wasteful evolutiona ry relic (Ogren, 1984; Heber et al., 1996; Wingler et al., 2000), while on the other hand, the pathwa y has been speculated to play roles in stress resistance, redox regul ation, and signaling (Oliveira et al., 2002; Slesack et al., 2007; Ahmad et al., 2008; Miller et al., 2009). To fully understand the issue

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3 of photorespiration, a basic comprehens ion of photosynthesi s must first be reviewed. By understanding plant bi ology, chemistry, and evolution, the passageway for greater study of the photorespirative pathway is built. 1.2 Photosynthesisan overview Photosynthesis is a biological oxid ation-reduction (redox) process which allows autotrophic organisms to use light energy to incorporate carbon dioxide into stable organic products. The overall reaction of this mechanism is stated as: Eq. 1.1 : CO2+ 2 H2A + hv (CH2O) + 2A + H2O For Rubisco oxygenic mechanisms, as o ccur in photosynthetic eukaryotes: Eq. 1.2 : CO2 + 2 H2O + hv (CH2O) + O2 + H2O where hv stands for light energy in both equations, and H2A is the generic reductant in Eq. 1.1 (Buchanan et al., 2000). In equation 1.2, water is split into hydrogen and oxygen by photon energy, and the electrons released are energized and ultimately transferred to NADP+ (nicotinamide adenine dinucleotide phosphate) yielding NADPH, which is ultimately used to reduce carbon (Buchanan et al., 2000; Heldt, 2005).

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4 Figure 1.2 : The basic structure of a chloropl ast, including the outer and inner membranes, the thylakoids, which are or ganized into grana, and the stroma. (Davidson, 2004) In photosynthesis, light is coll ected primarily by chlorophylls a and b pigments that are capable of absorbing li ght at wavelengths in the blue and red ranges of the visible light spectrum. Chlorophyll is found in the chloroplast, an energy-harvesting plastid. When light in the range of 500-570 nm shines upon a plant, it is reflected away. This range of wavelengths is perceived as the color green, which gives plants th eir verdant hues (Buchanan et al., 2000). Chlorophyll a has absorbance peaks at 430 and 662 nm, and constitutes the photosynthetic reaction centers in photosystems II and I, while chlorophyll b has absorbance peaks of 450 and 642 nm and surrounds the chlorophyll a reaction centers (Heldt, 2005). Chloroplasts from higher plants are surrounded by a double-membrane system consisting of an inner and outer envelope, as well as another internal

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5 membrane system composed of the thylakoid membranes (Buchanan et al., 2000). The inside of the chloroplast is divided into several different sections (Fig. 1.1): grana, which are stacks of the thylakoi d membrane pressed closely together; unstacked stromal thylakoid membranes; the internal thylakoid space known as the lumen; and the stroma, the fluid filling the space outside the thylakoids (McMurry and Begley, 2005). Chlorophy lls are embedded in the thylakoid membranes. The process of obtaining energized electrons leading to NADPH and ATP synthesis and the production of sugars can be broken up into two distinct parts, known as the light reactions (also known as the light-dependent reactions) and the light-independent reactions. The light reactio ns are responsible for the splitting of water molecules to produce oxygen and th e production of the reduced coenzyme NADPH. NADPH is then used in the li ght-independent reactions to reduce CO2 and produce sugar. The chemical equations of these two reac tions are written below (Buchanan et al., 2000): Eq. 1.3 : Summary of the light reactions: o 2H2O + 2 ADP + 2 Pi + 2 NADP+ + hv O2 + 2 ATP + 2 NADPH + 2 H+ Eq. 1.4 : The light-independent reaction: o 3 CO2+ 5 H2O + 6 NADPH + 9 ATP glyceraldehyde-3-phosphate (G3P) + 6 NADP+ + 3 H+ + 9 ADP + 9 Pi

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6 A portion of the G3P sugar is then diverted to sucrose and starch synthesis, as will be described in the subsequent sections. Both sets of reactions will be described in detail below. 1.3 The Light Reactions The light reactions occur in associatio n with the thylakoid membranes. As implied by the name, the light reac tions depend upon light to produce energetically useful molecules such as NADPH and ATP. The production of ATP and NADPH molecules is achieved by Photosystems II and I, the cytochrome b6f complex, the electron carriers plastoquinone and plastocyanin, and ATP synthase, as shown in figure 1.2. Figure 1.3 : The light reactions. Illustrated in sequence are photosystem II (PSII), plastoquinone (PQ), the cytochrome b6f complex, plastocyanin (PC), photosystem I (PSI), and ATP synthase (Taiz and Zeiger, 2002). In this series of reactions, light-har vesting complexes (LHCs) capture solar energy and channel it via resonance en ergy transfer into the photosystems’ reaction center chlorophy lls. The reaction center chlorophylls’ receipt of

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7 resonance energy allows them to transfer excited electrons to a series of electron carriers (Karp, 2010). By sending electrons through the electr on transport chain (ETC), energy is slowly released and used to create the ener getic molecules ATP and NADPH, the energy currency of the chloroplast. The reactions begin at photosystem II, an integral membrane protein complex which contains the P680 chlor ophyll reaction center and contains over 20 proteins per PSII unit at a 3 resolution (Komura et al., 2006). A molecule of water is oxidized via a manganese-calci um cluster (Karp, 2010) in the oxygen evolving complex, yielding 1/2 O2 and 2 H+. The electrons harvested from this reaction are funneled into the photosystem II reaction center. Photons excite the electrons in the PSII reaction center cau sing them to reach a higher energy level (Heldt, 2005). Upon excitation of the P680 center, one el ectron is released and transferred to pheophytin in the D1 pr otein, and then is transferred to plastoquinone, thus forming a semiqui none radical (Lack, 2005). Plastoquinone accepts two electrons and two prot ons, thereby being reduced to plastohydroquinone (a.k .a., plastiquinol). Plastohydroquinone then diffuses thr ough the thylakoid lip id bilayer and transfers its electrons to the cytochrome b6f complex. This integral membrane protein complex contains three electron car riers: cytochrome f, the Reiske Fe-S protein, and two different heme versions of cytochrome b6f (Lawlor, 2001). This complex then transfers the electrons to the copper-containing plastocyanin, which transfers the electrons over to PSI. Th e electron leaves photosystem I via the ferrodoxin protein, which is found on the st romal side of the membrane. During

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8 several of these step s, protons are taken from the stroma and released into the lumen. This trafficking of electrons produ ces an electrochemical gradient called the proton motive force (pmf). This force is capable of powering the final protein in this process, ATP synthase, also called the CF1–CF0 complex (Buchanan et al., 2000; Berg et al., 2007). ATP synthase evolved early in the course of life on ear th, and its basic structure is highly conserve d in bacteria, mitochondria, and chloroplasts (Heldt, 2005). The hydrophobic CF0 complex found in the thylakoid membrane conducts protons from the lumen into th e stroma, while the hydrophilic CF1 found in the stroma catalyzes the formation of ATP from ADP and Pi (Mauseth, 2009). CF0 consists of four different polypeptide ch ains, named I, II, III, and IV with corresponding masses of 17 kDa, 16 .5 kDa, 8 kDa, and 27 kDa and a stoichiometry of 1:2:12:1. This complex is similar in structure to the mitochondrial ATP synthesizing enzyme. Po lypeptide chains I and II coincide with polypeptide b from mitochondrial ATP synthase, while III corresponds to the C ring, and IV is similar in sequence to subunit a (Berg et al., 2007). The mechanism that powers ATP synthase is found in the CF0 subunit, and depends on polypeptide chains III and IV. Subunit IV is hypothesized to have 2 hydrophilic channels that do not completely span the thylakoid membrane. It is thought to be positioned in such a way th at each half-channel directly interacts with a subunit on III. Due to a negatively charged amino acid found in subunit IV, H+ can enter the channel connected to th e lumen. Because of the difference in electronegative charge between the 2 IV channels subunit III can rotate, carrying

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9 the hydrogen atom away from subunit IV and moving another H+ into the second channel connecting into the stroma. The H+ is then released from CF0 into the stroma, and helps to establish the pmf (Berg et al., 2007). This relative rotational movement of the subunits is used to power the formation of ATP. ADP and Pi enter the subunits from the 33 barrel, and the movement from the subunit connected to III subunit creates open (O), loose (L), and tight (T) conformations. O allows the input of ADP and Pi and facilitates the release of ATP; L binds ADP and Pi ; and T binds ADP and Pi with great avidity, promoting ATP formation (Berg et al., 2007). Maintaining the flow of electrons thr ough Photosystems II and I is essential for chloroplast protection ag ainst oxidative stress. Phot osystems can be damaged by excess energy in the form of sunlight via production of reactive oxygen species (ROS) (Foyer et al., 2009). When plants are under temperature-related stress, energy absorbed by the photosynthetic m echanism may be channeled to oxygen, and photooxidative radicals and other species can occur. In some cases, this is because the photosynthetic machinery ca nnot keep pace with the metabolic demand; for example, in cold temperatures, CO2 fixation slows down due to decreased enzyme activities, and some energy cannot be channeled to CO2 reduction (Rizhsky et al., 2003). Under drought conditions (water stress combined with high temperatures), the stomata close which limits gas exchange and prevents water loss (Heldt, 2005). This then leads to increased singlet oxygen and superoxide products (Foyer and Noct or, 2005). Excess excitation of the photosystems could result in excessive reduction of the components of the

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10 photosynthetic electron transport chain. Ma intaining electron flow down the ETC, even under the duress of stress, is thus vital to prevent damage to the plant (Rizhsky et al., 2003). Very high excitation of photosystem II, which is marked by an accumulation of plastohydroquinone, results in damage to the photosynthetic apparatus called photoinhibition (Taiz and Zeiger, 2002). Components most often damaged by excess energy in the chloroplast are D1 and D2 complexes, which are the core polypeptides of the PS II r eaction center, and are made up of over 20 proteins a piece (Buchanan et al., 2000). When the D1 complex, which has a high turnover even during normal photosynthetic conditions is damaged more quickly than the rate of its resynthesis, photoinhi bition occurs (Taiz and Zeiger, 2002). A number of different compounds and pa thways are thought to cooperate in protecting the photosystems mechanisms from photooxidative stress. Carotenoids, for example, are accessory pigments us ed to bring both ch lorophyll and oxygen atoms back to ground state. They do this by forming a triplet carotenoid molecule capable of dissipating energy in the form of heat (Nechushtai et al., 1988). The plant cell can also use a chloroplast avoi dance mechanism, in which chloroplasts move from the cell surface to the side walls in order to reduce light damage (Kasahara et al., 2002). Yet another system called the zeaxanthin cycle dissipates energy in the form of heat in what is called non-photoc hemical quenching. Zeaxanthin, which is synthesized from vi olaxanthin via the xanthophyll cycle, is directly responsible for protec ting antenna molecules (Buchanan et al., 2000; Rizhsky et al., 2003). The Water-Water cycle (a. k.a., the Mehler reaction) also

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11 assists with excess energy by producing superoxide radicals (O2 -) from the chloroplast. While this seems counterintu itive, the electric ally charged oxygen radical can then be reduced by the enzy me superoxide dismutase (SOD), turned into hydrogen peroxide (H2O2), and then further reduced by ascorbate peroxidase (APX) into two molecules of water (Heldt, 2005; Miller et al., 2009). 1.4 The Light-Independent Reactions The Light Independent Reactions were first elucidated in the 1950s by Melvin Calvin, Andrew Benson, and James A. Bassham (Calvin et al., 1951). The researchers used a kinetic approach to fi gure out the step-by-st ep process of this cycle. By applying 14CO2 to cell suspensions of green algae and taking samples at short intervals so they co uld identify the radiolabeled compounds, they were able to elucidate the intermediates. These re searchers were later awarded the Nobel Prize in Chemistry in 1961 fo r their ground-breaking work. ATP and NADPH formed during the light reactions are used to power the light independent reactions, as shown in figure 1.3. As the name connotes, this reaction does not directly depend on light. The cycle used to be referred to as the dark reactions, but since it doe s not occur solely at night and is paired up with the light reactions, it is now cal led the light independent reactions. Even the name “light independent” is somewhat misleading, since multiple enzymes in the

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12 Figure 1.4 : The Calvin Cycle. In this pict ure, RuBP reacts with of carbon dioxide to produce 3-phsophoglycerate (3 PGA). The 3PGA is converted to 1,3bisphosphoglycerate, and then glyceralde hyde-3-phosphate (G3P), which is used to produce sugars and starches. Most G3 P is then converted into ribulose-6phosphate and then RuBP to restart the cy cle, while a porti on is diverted for synthesis of sugars and other cellula r chemicals (Campbell and Reece, 2004).

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13 pathway are regulated by li ght, leading some researcher s to suggest changing the name again to the carbon reactions (B uchanan, 2000). This new designation has yet to be commonly utilized in literature and text, however. The light independent reactions, also collectively known as the CalvinBenson-Bassham cycle, reductive pentose phosphate cycle, or the C3 carbon fixation pathway, consists of three distin ct processes: carbon fixation, reduction reactions, and RuBP regeneration (Kar p, 2008). For every one molecule of C3 sugar phosphate produced, three molecules of CO2 are needed (Buchanan et. al, 2000). Refer to figure 1.3 for the following sections. 1.4.1 Carbon fixation In the beginning of the Calvin Cycle reaction, Rubisco fixes carbon dioxide by combining it with Ribulose 1,5 bisphosphate (RuBP). This enzyme will be discussed in more detail subsequently. The reaction it catalyzes produces 6 molecules of 3PGA (3-phosphoglycerate) for every three molecules of RuBP and CO2. Although 3PGA is the first stable produc t formed in the fixation of carbon dioxide, it is not the initial product. Inst ead, 3PGA forms in two concerted steps. The carboxylation of the C5 sugar RuBP produces a C6 intermediate that is immediately cleaved into two molecules of 3PGA (Buchanan et al., 2000). 1.4.2 Reduction reactions The 6 molecules 3PGA are turned in to 1, 3-bisphosphoglycerate with the input of 6 molecules of energy in the form of ATP and the enzyme

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14 phosphoglycerate kinase (Lack, 2005). W ith the addition of 6 molecules of NADPH and the enzyme glyceraldehyde -3-phosphate dehydrogenase, the 1, 3biphosphoglycerate is reduced to 6 GAP (glyceraldehyde 3-phosphate) molecules. One molecule of GAP leaves the Calvin Cycle and is used for biosynthesis and energy, while another five are regenera ted back into the light independent reactions with the assistance of ATP and returned as ribulo se-5 phosphate (Karp, 2010). 1.4.3 Regeneration Five GAP molecules are used to regenerate RuBP in a complex carbon shuffling mechanism. One of the five GAP molecules is isomerized by triosephosphate isomerase (TPI) into dihydroxyacetone phos phate (DHAP), which is then combined with a GAP molecule to form fructose 1,6-bisphosphate by the enzyme aldolase. Fructose 1,6-bisphospha te is dephosphorylated into fructose 6phosphate (F6P) via the enzyme Fructose 1,6-bisphosphatase. F6P and another GAP molecule are converte d by transketolase to form erythrose 4-phosphate and xylulose-5-phosphate (Xu5P). The enzyme aldolase catalyzes the formation of sedoheptulose 1,7-bisphosphate fr om erythrose 4-phosphate and dihydroxyacetone phosphate. Sedohe ptulose 1,7-bisphosphate is dephosphorylated into sedoheptulose7-phosphate (S7P) by sedoheptulose bisphosphatase. S7P and G3P is converted into xylulose 5-phosphate and ribose5-phosphate via a transketolase, and xyl ulose-5-phosphate is converted into ribulose 5-phosphate by phosphopentose epim erase. From here, all ribulose 5-

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15 phosphate needs to regenerate back into RuBP is three additional ATP molecules and the enzyme phosphoribulokinase (Buchanan et al., 2000; Taiz and Zeiger, 2006). The energetic requirements for the ne t yield of one GAP molecule from three fixed carbon dioxide molecules are nine molecules of ATP and six molecules of NADPH. Usi ng standard free energy changes for the hydrolysis of ATP and NADPH, creating one GAP molecu le is approximately 90% efficient (Buchanan et al., 2000). 1.5 Forms of Rubisco Ribulose 1,5 bisphosphate carboxyla te/oxygenase, a.k.a. RuBisCO or Rubisco, is the enzymatic protein which ma kes sugar synthesis possible. It is one of the few mechanisms capable of pr oducing reduced organic carbon from carbon dioxide (Tabita et al., 2007). So far, the scientific community only knows of four metabolic processes capable of using car bon dioxide as their single source of carbon: the Calvin-Benson-Bassham Cycle, the bacterial reduc tive tricarboxylic acid pathway, the Wood-Ljungdahl acetyl coenzyme A pathway (a.k.a., the reductive acetyl-CoA pathway) used by anaerobic microbes (Ragsdale and Pierce, 2008), and the hydroxypropionate pathway (Tabita et al., 2007). The lion’s share of this carbon fixation occu rs through the Calvin-Benson-Bassham cycle, which implements the enzyme Rubisc o for this purpose (Tabita et al., 2007). Rubisco is an ancient molecule, estimat ed to have first evolved about 3.8 billion years ago (Nisbet and Nisbet, 2008) At this early st age, the earth was

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16 inhospitable to any form of oxygen-respir ing life. During the time of Rubisco’s inception, there was little to no buildup of free oxygen in the atmosphere. It would be about another 2.4 gigaannum/b illion years (Ga) until oxygen became a significant percentage of atmospheric content (Buchanan et al., 2000; Buick, 2008). Over almost four billion years, R ubisco evolved into four unique forms: Rubisco I, the form found in cyanobact eria, algae, plants, and autotrophic proteobacteria; Rubisco II, the form f ound in anoxygenic photobacteria; Rubisco III, the form found in Chlorobacteriacae, a.k.a., green S bacteria; and Rubisco IV, the form found in methanotropic arch aea (Portis and Parry, 2007; Nisbet and Nisbet, 2008). Rubisco form IV is also known as a Rubisco-like protein (RLP), due to the fact that it is homologous to Rubisco form I’s large subunit. Despite the protein’s striking similarities to Rubisco form I, it does not carry out the processes of carbon or oxygen fixation. Instead, it ha s been found to function in other biological mechanisms, such as the methionine salvage pathway (Ashida et al., 2003; Tabita et al., 2008).

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17 Figure 1.5 : The four forms of R ubisco large subunits. C. tepidum is form IV, Spinach is form I, T. kodakarensis is form III, and R. rubrum is form II of Rubisco. All forms are composed of dimers of catalytic large subunits. Form I is the only version of Rubisco with small s ubunits (not shown in this figure), and has four dimers with small subunits f ound on the top and bottom of the octomeric core. Form II is comprised of between 1-8 large subunits. Form III, found only in some archea, is found with large subunits (L) in L2 or 5(L2) forms (Tabita et al. 2007). Rubisco I is the form commonly found in plants today. This specific form of Rubisco is found in only oxic organisms, and consists of eight large subunits and eight small subunits (Nisbet and Nisbet, 2009). With each large subunit weighing 51-58 kDa and each small subunit weighing between 12-18 kDa, Rubisco form I is one of th e largest proteins found in na ture with a to tal molecular weight between 504-608 kDa (Heldt, 2005). In plants, there is a division of labor for housing the genetic information to produce Rubisco I: the plastid genome encodes the large subunits, while the nuclear DNA codes for the small subunits (Taiz and Zeiger, 2002). While the large subunits are well established as the catalytic centers of the protein, the functi on of the small subunits is not certain. It has been proposed that the eight small subunits are used to stabilize the complex

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18 of the eight larger subunit s; however the bacteria Rhodospirillum rubrum throws a kink into that theory. This phototrophic purple bacterium utilizes Rubisco form II which only contains a dimer of the large subunit, yet its cata lytic properties are virtually the same as Rubisco form I (Tabita et al., 2007). While Rubisco I is currently the mo st common form of Rubisco in the biosphere, it is by no means the oldest. Rubisco form IV, utilized by methanotropic bacteria, is estimated to have evolved approximately 4 billion years ago (Nisbet and Nisbet, 2009). In comparison, Rubisco form I evolved an estimated 2.9 billion years ago, and ma rked the first time that oxygenesis had planetary impact. This period of time also coincides with the age of the oldest extant, large-scale carbonate reefs. Today’s such reefs depend on the photosynthetic organism zoozanthellae to produce oxygen for the coral animal, which suggests that the oxygenic photo synthetic mechanism may have been established. Oxygen is thought to have built up in the water, if not yet in the air (Nisbet and Nisbet, 2009). In the span of about a half billion years, oxygen built up in the atmosphere until the Great Oxid ation event (2.4 Ga), which marked a steep increase in oxygen content in the at mosphere. It was around that time that the first eukaryotic organisms were estim ated to have evolved and eventually brought about oxygen-respiring life as we know it (Nisbet and Nisbet, 2009). Rubisco is widely regarded as the most abundant protein on earth. Additionally, it accounts for as much as 50% of tota l water soluble proteins found in plant leaf tissue, and harbors an esti mated 30% of total nitrogen in leaves (Nishimura et al., 2008). Furthermore, the concentr ation of the catalytic subunits

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19 of Rubisco in chloroplast stroma can be as high as 4-10 x 10-3 mol/L. The staggering abundance of this behemoth of a protein may in part be explained by its slow catalytic turnover rate: at 3.3 molecules fixed by each subunit per second, it is one of the slowest enzy mes in nature (Spreitzer et al., 1993). Along with Rubisco’s large mass, as tounding numbers, and slow catalytic rate, this protein also has a sliding scal e of specificity for carbon dioxide versus oxygen. Factors such as temperature, light intensity, and atmospheric composition affect Rubisco’s ability to selectivel y fix carbon dioxide to produce organic compounds, versus reacting with oxygen in what many scientists view as a wasteful process, photorespiration. Befo re this pathway can be understood, Rubisco itself must be thoroughly described. 1.5.1 Rubisco activation and structure All forms of Rubisco are multimeric, and can consist of up to two types of subunits, the catalytically active large subun it and the small subunit (Andersson, 2008). Rubisco depends on a number of di fferent proteins for modulation of activity, control of activation, and medi ation to assure proper folding and assembly in the cell, though the specifics of these processes vary from different form and species containing the gene s to produce Rubisco. Despite the many differences found in amino acid sequence and function (e.g.: RLP), the secondary structure of the Rubisco large subunit has been highly conserved (Andersson, 2008; Andersson and Backlund 2008).

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20 In this thesis, the focus will be most specifically on Rubisco form I, the most common form found in oxic organisms, which as mentioned before consists of eight large and eight small subunits (Nisbet and Nisbet, 2009). The Rubisco form I enzyme has been further different iated into forms IA-ID, of which IA and IB occur in green organisms (cyanobacter ia, eukaryotic algae and green plants) and IC and ID are allotted to the so-c alled red-type enzymes (found in non-green algae and phototrophic bacteria) (Andersson and Backlund, 2008). The carboxytermini of the large subunits in every Rubisco has been found to have the same conserved structure: a seque nce of eight consecutive units, each made up of about 200 amino acids that are ar ranged into an eight-stranded / barrel (Spreitzer and Salvucci, 2002; Ster ner and Hocker, 2005; Andersson and Backlund, 2008). This ( )8 barrel template has been found in various enzymes in nature, and facilitates five out of six known enzymatic reactions as defined by the Enzyme Commission: those catalyzed by oxi doreductases, transferases, lyases, hydrolases, and isomerases (Ste rner and Hocker, 2005). The ( )8 barrel structure was first found in triose phosphate isomer ase, a highly catalytically efficient enzyme found in the glycolytic cycle, and was therefore named the TIM barrel after the enzyme (Sterner and Hocker, 2005). Catalytic activity of Rubisco is regulated by the enzyme Rubisco activase (RCA). RCA, a nuclear-encoded chloropl ast protein (Zhang and Portis, 1999), facilitates activation and maintains Rubi sco’s catalytic activ ity. By using ATP hydrolysis to release tightly bound compounds from the active site of the enzyme, it regulates catalytic function (Kumar et al., 2009). Rubisco activase can remove

PAGE 27

21 the inhibitory sugar phosphate molecules, RuBP, and (in some plants) CABP (a Rubisco inhibitor) (Zhang and Portis, 1999). Although somewhat counterintuitive, the substrate RuBP can also act as an inhibitory molecule by binding to the inactive form of Rubisco and preventing carbamylation of the lysine by an outside CO2 molecule (Zhang and Portis, 1999). Part of the AAA+ family (ATPases associated with diverse cellular activi ties), RCA is generally present in a multimeric complex composed of two isoforms of different molecular mass (Kumar et al., 2009). The two isoforms differ only at their carboxy-termini, which for the most part only arises by alternativ e splicing of the gene transcript (Zhang and Portis, 1999). However in some species the two isoforms are products of two distinct genes (Kumar et al., 2009). The loop connecting -strand 6 with -helix 6 in the TIM structure has been found to be significant for Rubi sco specificity and catalysis (Tabita et al., 2007; Andersson and Backlund, 2008). Residues predominantly found between strands and -helices interact with the transi tion state analog 2-carboxy arabitinol 1,5 bisphosphate (CABP) (Spreitzer and Salvucci, 2002). Loop 6 has been demonstrated to partition between the opened and closed conformations of the enzyme by folding over the active site (Tabita et al., 2007). In order for the Rubisco to function, it also requires the assistance of a magnesium ion complexed to the enzyme. In closed conformation, the magnesium ion complexes with a glutamatic acid re sidue, an aspartatic acid residue, and a carbamylated lysine as shown in fi gure 1.5 (Sterner and Hocker, 2005).

PAGE 28

22 Figure 1.6 : The Rubisco active site. (Sterner and Hocker, 2005). 1.5.2 Rubisco reaction mechanism While there is a wealth of inform ation about the Rubisco catalytic mechanism, it is still not yet fully under stood. In order for Rubisco to function, it must first be activated by carbamy lation of a lysine residue by a CO2 molecule distinct from the CO2 in the active site (Anders son, 2008) followed by binding of an Mg2+ ion (Peterhansel, Neissen, and Keibesh, 2008). From there, the carbamylated Rubisco and magnesium ion can bind RuBP and convert it into the singly deprotonated 2,3-enedi ol form needed for initiation of the mechanism (Spreitzer and Salvucci, 2002). Once RuBP is bound, loop 6 of the carboxyterminal / barrel (or TIM barrel) extends ove r the active site, sealing it away from the solvent in the environment (Kannappan and Gready, 2008). The double bond from the enolate RuBP can then act as a nucleophile an d attack the other reactant (CO2 or O2) in an addition reaction.

PAGE 29

23 The carboxylation and oxygenation reactions, illustrated in figure 1.6, have multiple steps and at least three transition states (Andersson, 2008). For the carboxylation reaction, the sequence of reactions is 1) enolation of RuBP into the enediol intermediate, 2) car boxylation of the 2,3-enediola te, 3) hydration of the 2carboxy 3-ketoarabitinol 1.5 bisphosphate (C KABP) ketone intermediate into a geminal diol, 4) carbon-carbon scission between the C2 and C3 carbon, and 5) stereospecific protona tion of the resulting carbanion fr om the previous scission to produce two molecules of 3PGA (Spreitz er and Salvucci, 2002; Andersson, 2008; Kannappan and Gready, 2008). The oxygenation mechanism for Rubisc o is less well studied, but is assumed to use the same general mechanism and residues as the carboxylation reaction. Like the carboxylation reacti on, the oxygenation reaction has multiple steps: 1) RuBP must be converted in to the enediol form in order for the mechanism to begin 2) the nucleophil ic double bond from the RuBP enediol attacks an oxygen atom from the oxygen molecule, producing 2-peroxo-3-keto-Darabitinol 1,5-bisphosphate (PKABP). 3) The resulting ketone is hydrolyzed into a geminal diol, and 4) the C2-C3 bond is broken to produce one molecule of the metabolically useful product 3PGA, and one molecule of the photorespiratory chemical 2PG (Spreitzer and Salvucci, 2002).

PAGE 30

24 Figure 1.7 : The ribulose 1,5 phosphate car boxylase/oxygenase mechansisms. Before RuBP can be used in the carb oxylation or oxygenation mechanisms, it first has to be converted into its enedio late form (Kannappan and Gready, 2008). 1.6 Photorespirationoverview Photorespiration is the pathway necessitated by the oxygenation reaction catalyzed by Rubisco. The inhibition of carbon dioxide fixation in the presence of oxygen was first observed in 1920 by the N obel Laureate Otto Warburg; his discovery was known as the “Warburg E ffect” (Eckardt, 2005). This strange phenomenon was not fully understood until 1971, when N.E. Tolbert and various other scientists began to decipher th e mechanism of the Warburg Effect. Compiling the cumulative information, the Warburg Effect is the consequence of the oxygenation of RuBP catal yzed by Rubisco (Wingler et al., 2000). Due to Rubisco’s capability to catalyze RuBP’s reaction with either CO2 or O2, the two metabolic pathways compete with each othe r. The ratio of the carboxylation rate to the oxidation rate is dependent on the CO2 and O2 concentrations in

PAGE 31

25 environmental mediums such as water or air (Wingler et al., 2000). The ratio of carboxylation to oxygenation for these reactio ns are summed up in a formula: Eq. 1.5 : 2 2O CO K V K V v vc o o c o c c defines the maximum carboxylation rate, o defines the maximum oxygenation rate, Vc Ko / Vo Kc defines the specificity factor of Rubisco, and Kc and Ko are the corresponding Michaelis -Menten gas constants for CO2 and O2 (Wingler et al., 2000). Increasing temperature redu ces the specificity of Rubisco to CO2 due to enzyme kinetics and activation st ate as well as the concentration of dissolved CO2 in the cell, so photosynthesis has been found to be most productive in the early morning and least pr oductive in the afternoon (Tolbert et al., 1995). While the reaction with CO2 usefully produces two molecules of 3PGA which can enter the C3 Calvin cycle, the oxygenation of RuBP produces one molecule of 3PGA and one molecule of the two carbon molecule phosphoglycolate. Phosphoglycolate is reduced to the toxic molecule glycolate, which has to be sent out of the chlo roplast and broken dow n by a series of seemingly energetically wasteful proce sses detailed below and which ultimately reduce the overall productivity of the plant. However, is photorespiration a truly wasteful process? This thes is will discuss how photorespiration may play a role in stress resistance, redox regulation, and si gnaling, as well as possibly contributing to the regulation of earth’s climate and environment. The photorespiratory reacti ons reclaim 75% of the lo st carbon into useful products for the plant, howeve r it loses the other 25% as CO2 (Buchanan et al.,

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26 2000; Wingler et al., 2000). The pathway circuits th e chloroplast, the peroxisome, and the mitochondrion of the cell. Figure 1.8 : The photorespiratory reactions. In th is picture, we are concentrating on the photorespiratory cycle indicated in red. In black are the Calvin cycle and the Krebs cycle. Each photorespiratory enzyme is denoted with a number in the drawing, going from 1-13. Protein #1 is R ubisco, and has already been discussed in Ch. 1. Protein #2 is 2-phosphoglycolate phosphatase (PGLP); #3 is glycolate oxidase (GLO); #4 is serine:glyoxylat e aminotransferase (SGAT); #5 is glutamate:glyoxylate aminotransferase ( GGAT); #6 is glycolate decarboxylase (GDC); #7 is serine hydroxymenthyltran sferase (SHMT); #8 is hydroxypyruvate reductase (HPR); #9 is glycerate kinase (GLYK); #10 is glutamine synthetase (GS); #11 is glutamate synthetase (G LU/Fd-GOGAT); #12 are the dicarboxylate transporters (DIT); #13 is catalase (CAT ). PETC and RETC are respectively the photosynthetic and respiratory elec tron transport chains (Foyer et al. 2009).

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27 The photorespiratory cycl e occurs in the following way, as illustrated in figure 1.7. As previously mentioned, R ubisco catalyzes the reaction of 2 molecules of RuBP with oxygen to form three molecules of PGA and two molecules of glycolate-2-P. Glycolate-2-P is dephospor ylated to glycolate by a chloroplastic phosphoglycolate phosphatase (P GLP). Because glycolate is a toxic chemical to the plant, it is immediately removed from the stroma and sent into the peroxisome for degradation (Buchanan et al., 2000; Wingler et al., 2000; Heldt, 2005). After transport into the peroxisomes, glycolate is oxidized into glyoxylate by the enzyme glycolate oxidase (GLO). The glyoxylate is then transaminated (an amino group is transferred) to glycin e by serine:glyoxylate aminotransferase (SGAT) or by glutamine:glyoxylate am inotransferase (GGAT) (Wingler et al., 2000). Half of the glycine molecules are then converted to N5, N10-methylene tetrahydrofolate (THF) by the enzyme glycine decarboxylase (GDC) in the mitochondrion producing the useful chemicals CO2 and NH3 (Buchanan et al., 2000). The other half of the glycine molecules can react with THF in what is known as the serine hydroxymethyl-transfera se reaction (SHMT) to form serine. The serine is then transferred back into the peroxisome where it is converted by SGAT into hydroxypyruvate, and then hydroxy pyruvate is reduced to glycerate by hydroxypyruvate reductase (HPR) (Buchanan et al., 2000; Wingler et al., 2000). The glycerate is then returned to the chlor oplast and phosphorylated by glycerate kinase into gly cerate-3 phosphate, which is then converted to three

PAGE 34

28 3PGA and sent back into the Calvin Cycle. Due to the fact that NH3 is produced in this reaction, photorespiration is closel y linked to plant nitrogen metabolism, as will be discussed later in this thesis (Heldt, 2005). Because the process of photorespi ration expends energy to reclaim CO2, and some CO2 is lost to the atmosphere, a num ber of scientists describe the process as very wasteful (Spreitzer and Salvucci, 2002; Ellis, 2010; Liu et al., 2010). In total, 3.25 mols of ATP are used per one oxidative cycle (Wingler et al., 2000). Indeed, there is a good amount of research dedicated to reducing or completely removing photorespiration from plants in order to make them more efficient. Specific examples wi ll be discussed in chapter 3. 1.7 Alternate strategies for photosynthesis Due to variations in temperatur e, light intensity, and atmospheric composition, some plants evolved sligh tly different strategies to minimize photorespiration. The two alternate strategies are known as the C4 Cycle and the Crassulacean Acid Metabolism (CAM). 1.7.1 C4 metabolism C4 Photosynthesis likely developed from two environmental pressures: a decrease in atmospheric CO2 levels such that the gas became a significant limiting factor, and a concurrent warming of the earth’s atmosphere (Tolbert and Preiss, 1994). Because of these stressors, C4 plants developed a very effective mechanism for modifying their internal CO2 levels in both gas and aqueous

PAGE 35

29 environments. Because of this evolutionary advantage, C4 plants tend to fare better in geographical areas with a warm er climate (Tolbert and Preiss, 1994). Figure 1.9: The C4 Kranz anatomy. CO2 is fixed into a four carbon acid in mesophyll cells and then shuttled into bundl e sheath cells as a four carbon acid (in this diagram, malate). CO2 is released into the bu ndle sheath cell producing an area of concentrated carbon near Rubisc o and the Calvin cycle (Mallery, 2010). The C4 photosynthetic pathway was identi fied in the 1960s by Roger C. Slack and Marshall D. Hatch (Slack and Hatch, 1967; Hatch, 2002). When they found that when several plants species were supplied with 14CO2, instead of generating 3PGA as the first stable phot osynthetic intermediate, they produced large amounts of a four-carbon organic ac id later found to be oxaloacetate (as reviewed in Tolbert and Preiss, 1994). Upon further study of these plants anatomies, Hatch and Slack found two diffe rent types of chloroplast containingcells: mesophyll cells, and closely as sociated vascular bundles dubbed bundle sheath cells. This peculiar framework wa s later dubbed Kranz anatomy (Kranz is

PAGE 36

30 German for ‘wreath’, which describes what the bundle ce lls look like under microscope) (Buchanan et al., 2000). This Kranz anat omy is vital to the biochemistry of CO2 fixation in C4 plants. In C4 photosynthesis, CO2 is converted into HCO3 in the mesophyll cell by carbonic anhydrase (Hatch and Burnell, 1990 ). It is fixed into oxaloacetate and then into an organic acid (such as malate or aspartate, depending on the species) and transferred over to the bundl e sheath cells. There, the CO2 is released, causing a build-up of CO2 in the bundle sheath. At the resulting high concentration of CO2, Rubisco can catalyze the carboxylat ion mechanism versus the oxygenation mechanism, even in circum stances with high heat a nd intense sunlight. The C4 photosynthesis also helps plants conserve water by up to 50% compared to that of C3 photosynthesis (Heldt, 2005). 1.7.2 CAM Photosynthesis For plants growing in extremely dry and hot environments, C4 photosynthesis is no longer enough to ensu re survival. Succulents, which are named after their fleshy, water-retaini ng leaves, have evolved yet another photosynthetic pathway which allows them to survive in harsh environments. This series of reactions is known as the Cr assulacean Acid Metabolism (CAM). The name may seem to suggest that only plants of the family Crassulaceae have evolved such a pathway, this is not th e case. There are at least 18 documented flowering plant families that utilize the CAM photosynthetic cycle. In fact, it has

PAGE 37

31 also been found that some nonsucculent pl ants also use the CAM strategy (Kluge, M. and Ting, I., 1978). Crassulacean Acid Metabolism uses much of the same scheme of the C4 photosynthetic pathway in that it converts CO2 into HCO3 and fixes it into an organic acid (which is typically malate). CAM plants lack the Kranz anatomy and instead convert carbon dioxide into an organic acid at night, while CO2 is released near Rubisco during the day. In order to prevent water loss, CAM plants carry out CO2 uptake only during the nighttime, and keep their stomata closed during the day. By following this process, CAM plants are able to survive in extreme climates by minimizing water loss. 1.8 Conclusions While an understanding of these i ndividual mechanisms are useful, they do not provide a full depiction of how they interact with metabolism of the plant as a whole. With the preceding pathways in mind however, a further study of the photorespirative pathway and its potentia l benefits and detriments can be accomplished.

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32Chapter 2. The C2 pathway and relevant mutants 2.1 Introduction The photorespiratory pathway is rega rded by some scientists as highly inefficient: with an estimated carbon loss of 17-25% (Sharkey, 1988; Cegelski and Shaefer 2006; Nunes-Nesi et al., 2007) and an energetic cost of 3.25 mols ATP and 2 mols NADPH per one oxygenati on, there is reason to question the pathway’s efficiency (Wingler et al., 2000). However, these numbers do not take into account the numerous benefits of the photorespiratory system for the plant. Data suggest that the photorespiratory pathway is useful in a number of bio tic and abiotic conditions. Photorespiration has been shown to protect the plants in s ituations such as drought (water stress), high light intensity, extreme temperatures salt stress, and nitrogen reuptake (Wingler et al., 2000; Ahmad et al., 2008). This chapter will di scuss in detail the many mechanisms and protective features th at are directly or indirectly a product of photorespiration. 2.2 The effects of suppressing photorespiration The most immediate way to prove the essentiality of photorespiration is by seeing what happens when one down -regulates the pathway. To suppress photorespiration, plants can be housed in an environment with low oxygen content (LO). This LO atmosphere lead s to a temporary increase of the net photosynthetic rate (Pr); however, this increased rate does not last during

PAGE 39

33 prolonged incubation periods (Migge et al., 1999). After three days, the ultrastructure of the chlo roplast undergoes structural changes, the number of starch grains increase, and osmiophilic gl obules appear. These structural changes are reminiscent of those seen in leaf senescence (Schwabe and Kulkarni, 1987). Plant morphology also has been seen to change in plants grown under LO conditions versus those grown in ambient atmosphere. Plants exposed to LO were significantly smaller and thicker than the control plants (Migge et al., 1999), and have been shown to have reduced seed production (Musgrave and Strain 1988). The altered morphology of the LO leaves has been suggested to result in a reduction of transpiration, and thus wa ter loss (Musgrave and Strain, 1988). Ogren (1984) noted that studies whic h impaired the photorespiratory cycle under ambient CO2 produced symptoms of light stress in plants, such as photoinhibition and chlorosis. Photoinhibiti on occurs in conditions where the rate of photodamage exceeds the rate of repair. In a study by Takahashi et al., (2007), they found that impairment of the photores piratory system acceler ated the rate of photodamage, and thus spurred on ph otoinhibition, by inhibiting the de novo synthesis of the D1 protein (found in photosystem II) at the translation step (Takahashi et al., 2007). 2.3 Photorespiratory mutants To further study the significance of p hotorespiration in the plant cell, there have been numerous studies that supp ressed, overexpressed, or knocked out genes responsible for producing photorespirato ry enzymes. The photorespiratory

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34 pathway consists of 16 enzymes and more than 5 translocators spread across the chloroplast, peroxisome, and mitochondrion (Douce et al., 2001). Some of these photorespiratory enzymes have been studied with intensive deta il; however others have yet to be thoroughly covered. This section will modestly cover the forays into manipulation and study of these proteins. Starting from enzyme #2 (2phosphoglycolate phosphatase) to enzyme # 13 (catalase), each enzyme will be sequentially covered One interesting fact to keep in mind while reading this section is that deletion of any of the core enzymes of th e photorespiratory cycle results in severe sensitivity of the respective mutants to typical atmospheric conditions (Timm et al., 2008), as will be discussed shortly. This fact, above all others, suggests that photorespiration plays a vital role in pl ant biology. For reference throughout the discussion of these enzymes, refer to figure 1.7. 2.3.1 Carbon recycling photorespiratory enzymes 2.3.1.1 Phosphoglycolate phosphatase The chloroplastic enzyme phosphoglycolate phosphatase (PGLP) is the first enzyme in photorespira tion after the fixing of O2 by Rubisco. It reacts with the substrate 2-phosphoglycolate (2PG) to produce glyoxylate, which is then transferred into the peroxisome to react with glycolate oxidase (GLO). PGLP is a light-inducible and light-regulated enzyme that has been found in higher plants and green algae (Belanger and Ogren, 1987; Baldy et al., 1989). The enzyme specifically reacts only w ith 2PG, and requires Cland Mg2+ to properly function

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35 (Christeller and Tolbert, 1978; Seal a nd Rose, 1987; Schwarte and Bauwe, 2007). The molecular mass of PGLP varies in higher plants from 21-32 kDa, and is suggested to display both homodime ric and homotetrameric conformations (Hardy and Baldy 1986; Belanger and Ogren, 1987). The chemical 2PG is poisonous to plan ts, and in large quantities can result in many negative effects to their metabolism. For example, 2PG has been found to inhibit the Calvin cycle enzyme trios phosphate isomerase and the glycolytic enzyme phosphofructokinase (Anderson 1971; Kelly and Latzko, 1976; Schwarte and Bauwe, 2007). The gene(s) encoding PGLP were onl y somewhat recently discovered in 2007 by Sandra Schwarte and Hermann Bauwe, and thus have been of limited study. In Schwarte and Bauwe’s experime nt (Schwarte and Bauwe, 2007), they isolated two genes from Arabidopsis thaliana thought to code for the photorespiratory enzyme PGLP, and produ ced homozygous mutant lines to see whether either gene controlled PGLP production. They found that both genes coded for functional PGLP enzymes, however only one of them produced a phenotype suggesting stunted photorespi ratory action. The gene AtPGLP1 displayed the common charac teristics of poor growth (chlorosis, death) in a normal atmospheric environment compar ed to WT, AtPGLP1 mutants could survive in an increased (0.3%) CO2 environment, and was only able to seed in greatly increased (0.9%) CO2 conditions. The second gene encoding for PGLP, AtPGLP2, had seemingly no effect on plant growth or PGLP activity. AtPGLP2 was found to encode for a cytosolic

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36 version of PGLP, one that is most proba bly not utilized by the photorespiratory system due to the fact that leakage of 2P G from the chloroplast is unlikely. Thus, Shwarte and Bauwe theorized that AtPG LP2 is most likely related to the metabolism of minor amounts of 2PG from other processes in the cell (Shwarte and Bauwe, 2007). In another study by Eisenhut et al., (2008), the 2PG pathway in cyanobacteria was investigated. While th e conversion of 2PG is necessary for higher plants, it was previously thought to be superfluous for cyanobacteria due to their carbon concentratin g mechanism (CCM), in which, similar to C4 plants, they can catalyze the accumulation of CO2 around Rubisco (Raven et al ., 2008). In a previous study by Eisenhut et al (2006), they found evidence of a combined plant-like C2 and bacterial-like glycerat e pathway (figure 2.2) that metabolized 2PG in Synechocystis a strain of cyanobacteria. They targeted three genes for mutation: odc, which codes for oxalate decarboxylase in the decarboxylation pathway; gcvT, which c odes for a T-protein on the glycine decarboxylase (GDC) complex in the C2 pathway; and tsr which codes for tartronic semialdehyde reductase in th e glycerate pathway. The single and double mutants were capable of survival in no rmal atmospheric conditions. However, the triple mutant strain necessitated a hi gh carbon requiring environment (HCR). This is the typical phenotype of a C3 photorespiratory mutant, which is unexpected. Due to the fact that they have a CCM, the common conception was that cyanobacteria should produce little to no 2PG and thus would never display a C3like photorespiratory phenotype.

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37 Figure 2.1 : The 2PG metabolism in Synechoc ytsis sp. Strain PCC 6803. Notice the similar photorespiratory enzymes (PGL P, GDC, SHMT, HPR, GK), as well as some of the differences (glycolate de hydrogenase takes the place of GLO, there are nonspecific aminotransferases, un like SGAT and GGAT, no GS or GLU). Two genes (glc01, glc02) encode for th e enzyme glycolate dehydrogenase to produce glyoxylate. The gene odc codes for oxalate decarboxylase, the gcvT gene codes for the T-protein of the GDC, a nd the tsr gene codes for tartronic semialdehyde reductase (Eisenhut et al. 2008). Interestingly enough, the HCR character istic appearing in the triple mutants was lost after several cycles of cultivation in normal atmospheric CO2 conditions, suggesting that the cyanobacter ium eventually overcame the issue of 2PG (Eisenhut et al., 2008). Finally, this triple mutant photorespira tory phenotype suggests that 2PG is still produced despite the CCM, and may be necessary for healthy growth in cyanobacteria as well as C3 plants in the current atmospheric environment. Further evidence pointing towards this cl aim has also been identified. 2PG has been shown to serve as a signal for cyanobacteria to acclimate to normal

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38 atmospheric CO2 levels when moved from high CO2 levels (Nishimura et al., 2008; Eisenhut et al., 2008), a process that may be similar to a C3 plants’ ability to use potentially toxic chemical byproduc ts such as ROS for stress signaling. This speculation is further back ed by work done by Hackenberg et al., (2009) who found that Synechocystis mutants with knocked out gene sequences encoding GDC T-protein and flavoproteins (used in the Mehler reaction to cope with ROS) aid under the stresso r of high light. Additionally, the enzymes necessary for the plant-like C2 cycle are present in all currently known complete ge nome sequences, including marine Prochlorococcus and Synechococcus strains, which are thought to only possess minimal genomes containing only genes necessary for survival in their environment (Dufresne et al., 2003; Eisenhut et al., 2008). All of this evidence together suggests that some form of photorespiratory reactions have been occurring for far longer than we had previously expected. 2.3.1.2 Glycolate oxidase Glycolate oxidase (GLO), is one such enzyme found in the photorespiratory mechanism. GLO is an oc tomeric protein, with each identical 40 kDa subunit containing a / barrel motif corresponding to a flavin mononucleotide domain (FMN) (Douce and Neuberger, 1999). Located in the peroxisome, this protein catalyzes the irreversible reaction of glycolate and oxygen into glyoxylate a nd hydrogen peroxide (H2O2). This reaction can be divided into two half reactions : 1) glycolate is oxidized by a flavin buried deep in

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39 the barrel, and 2) FM N is reoxidized by O2 to produce H2O2 (Douce and Neuburger, 1999). As GLO activity proceeds over the co urse of a day, large amounts of H2O2 are released into the peroxisome (Douce et al., 2001 b). Some of the H2O2 can be degraded into oxygen and water by the en zyme catalase, but the high MichaelisMenten constant ( m) for the enzyme results in some H2O2 diffusing to the inner surface of the limiting peroxisomal membrane, which contains ascorbate peroxidase (APX), an enzyme in the ascorbate-glutathione cycle (Douce and Neuburger, 1999; Nunes-Nesi et al., 2008). The ascorbate-gl utathione cycle will be discussed in further detail at a later point in this paper. In a study by Xu et al., (2009), the team produced a novel transgenic strain of rice ( Oryza sativa L.) which carries a GLO antisense gene driven by an estradiol-inducible promoter. This constr uct allowed them to suppress GLO in a controlled manner. Transgenic plants tr eated with estradiol showed severely stunted growth, a typical phot orespiration-deficient pheno type. When faced with a 30%, 60%, and 90% reduction of GLO ac tivity, there accumulated a respective 40, 60, and 130-fold increase in the toxic chemical glycolate. However, other downstream metabolites, such as glycine and serine were not reduced under GLO deficiency. This suggests that while GL O is the major pathway to metabolize glycolate, there may be an alternativ e pathway for the production and regulation of glyoxylate (Xu et al., 2009). At a 60% reduction of GLO activity, the photosynthetic rate (PN) was reduced, and PN was found to linearly decline wi th GLO above 60% reduction of

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40 the enzyme’s activity. It was also f ound that the maximum efficiency of photochemistry after dark adaptation (expressed as Fv/Fm, where Fv stands for the maximum capacity for photochemical quenching, and Fm stands for the maximum chlorophyll fluorescence value) remained constant at first, and then dropped sharply after ~ 60% suppression of GLO activities. A similar pattern was observed for nonphotochemical quenchers (NPQ) (Xu et al., 2009). This positive and linear correla tion between GLO activity and PN suggests that GLO can exert regulati on over photosynthesis. Xu et al. (2009) also discovered that a number of genes in the GLO mutants were expressed much differently than the wild type. A gene expressing 2-phosphogylcolate phosphatase (PGLP) was also suppressed, while isocit rate lyase (ICL) and malate synthase (MLS) were highly upregulated. Furthe rmore, Rubisco activase (RCA), an enzyme that regulates Rubisco activity, was also suppressed. Since suppression of GLO produces an effect that simulates stressed plants, I conjecture that GLO’s regulati on of photosynthesis may have to do with the plant’s stress response. Most stresses (l isted in chapter 4) re sult in a surplus of excited electrons, which create reactiv e oxygen species (ROS), and these ROS have been implicated in a complicat ed system of cell signaling. When Fv/Fm and RCA are downregulated synchronously with PN, there may be a ROS-regulated signal transduction pathway which reduces activity of PSII and Rubisco in response to stress. I would like to see studies of the ROS levels of the GLO mutants, particularly in the amounts of ROS produced in the chloroplasts and peroxisomes. Further study of the me chanisms controlling PSII activity, RCA

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41 activity, and ROS in the chloroplast and pe roxisome would be a very interesting foray into plant biochemistry. Contrasting to Xu et al.’s work, Fahnenstich et al., (2008) produced a transgenic line of Arabidopsis thaliana plants which overexpressed GLO. These transgenic plants displayed between 20-50% higher GLO activities than the wild type (WT). They found that homozygous GLO mutants growing at moderate photon fluxes of 75 mol quanta m-2 s-1 in ambient CO2 concentrations were smaller than the WT, and presented rosett es with a paler gree n color and reduced diameter. Overall, the GLO plants displa yed retarded growth and flowering time. At the same time, the level of glyoxylate and H2O2 was unsurprisingly increased, though the increase in H2O2 was observed only after exposure to 200 mol quanta m-2 s-1. Photosynthetic electron transport ra te (ETR) markedly decreased, while Fv/Fm displayed no difference between mutant and WT (Fahnenstich et al., 2008). Overall, Fahnenstich et al. (2008) hypothesized that increased levels of H2O2 may have affected transcription f actors involved in plant growth and flowering. GLO-overexpressing plants al so interestingly displayed reduced expression of genes involved in anthocya nin biosynthesis. An thocyanin is shown to act as a “sunscreen” by absorbing blue -green light, and thus protecting plants from photoinhibition (Vanderauwera et al., 2005). The low levels of anthocyanins most likely also contributed to th e GLO overexpressors’ stunted growth. Fahnenstich noted that Vanderauwera et al., (2005) had recently found a transcriptional cluster including anth ocyanin biosynthetic and regulatory

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42 pathways which were strongly induced by high light in WT, but delayed in catalase (CAT)-deficient plants. Taking Xu et al., (2009), Fahnenstich et al., (2008), and Vanderauwera et al.’s (2005) work together, there is some evidence to suggest that perhaps photorespiration may be related to the ex tent of ROS signaling, but much more research needs to be done. 2.3.1.3 Glutamate and Serine -g lyoxylate aminotransferase The enzymes next in line for discussion are glutamate:glyoxylate aminotransferase (GGAT) and serine:glyoxylate am inotransferase (SGAT). GGAT catalyzes the reaction of glutam ate and glyoxylate (GLO’s product) to produce 2-oxoglutarate and glycine. Seri ne glyoxylate animotranferase (SGAT), on the other hand, reacts with serine produced from another photorespiratory enzyme, serine hydroxymethyltransfer ase (SHMT), to produce hydroxypyruvate. Both enzymes are found in the peroxisome. Between both GGAT and SGAT, these two proteins are thought to play a major role in the synthesis of major amino acids, nitrogen recycling, and citrulline production in the plant cell (Igarashi et al., 2006). The photorespiratory reactions have been found to contribute to the formation of glutamine (gln), glutamate (g lu), serine (ser), and glycine (gly) due to the action of GGAT and SGAT (Foyer et al., 2009). Serine and glycine, in particular, are very important and serve as precursors in a number of biological pathways, such as phospholipid synthe sis (which uses serine) and purine

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43 formation (which uses glycine). Serine and glycine can be synthesized through one other pathway other than photorespi ration in photosynthetic cells. During glycolysis, serine is formed from 3phosphoglycerate, a chemical found in the Calvin cycle. Glycine on the other hand is obtained from serine through a serine hydroxymethyltransferase (SHMT)-catalyzed reaction. Despite the fact that there are other pathways for the synthesis of glycine and serine, photorespiration has been suggested to be the major pathway for the formation of these amino acids because of the high flux of carbon through this cycle (Gerbaud and Andre, 1979; Igarashi et al., 2006). Leaf mitochondria are capable of rege nerating ammonium at as much as 50 times the rate of nitrogen reductio n via glutamine synthetase (GS2) and ferredoxin-dependent glutamate synthase (Fd-GOGAT), and the ammonium can then be used for plant growth. Since GGAT supplies the substrate of glycine, which is then broken down into NH3 and CO2 via GDC, this enzyme provides a major contribution to the nitrogen re assimilation cycle. At elevated CO2 conditions, photorespiration is limited and has been found to limit the assimilation of nitrogen in the shoots of dicotyled ons and monocotyledons, suggesting that photorespiration is necessary for the effective use of nitrogen (Bloom et al., 2002; Rachmilevitch et al., 2004; Igarashi et al., 2007). In a study by Igarashi et al., (2007) 42 mutant lines overexpressing GGAT were created for study. In this experime nt, the authors found that all 42 mutants displayed increased levels of glycine, se rine, and citrulline. One of their lines accumulated citrulline, serine and glycine at 13.4, 3.2, and 2.4 times the rate

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44 found in WT plants under normal atmospheric conditions, and 5.2, 1.9, and 1.9 times the rate found in an increased CO2 environment. Furthermore, the knockout GGAT mutant displayed no accumulation of serine in either environment, suggesting that GGAT is responsible for most of the synthesis of serine. In another study by Verslues et al., (2007), they screened for mutant plants with decreased RD29A:LUC expressi on (a marker gene responsive to desiccation) and were able to identify tw o mutant alleles of GGAT that displayed reduced abscisic acid (ABA) sensitivity. ABA is a key component in plant stress response, particularly from stressors th at produce a drop in tis sue water content. The authors found that the GGAT mutant ca used a light-dependent increase in H2O2, which is consistent with the fact th at GGAT plays a role in photorespiration reactions in the peroxisome. This increase in H2O2 caused a change in the basal level of H2O2 in unstressed plants, which then in turn caused an increase in proline accumulation, altered ABA-induced gene expression, and reduced ABA accumulation. The increased levels of prolin e were especially in teresting in light of evidence suggesting that proline meta bolism is connected to redox regulation and may by itself serve as an antioxidant (Hare et al., 1998; Verslues et al. 2007). Furthermore, while the stress-i nduced accumulation of proline is dependent upon ABA, the application of ABA to an unstressed plant does not elicit the same level of pro line accumulation as seen in stressed plants (Verslues and Bray 2006). In Verslues et al. ’s (2007) work, they found that their GGAT mutants displayed higher levels of prolin e accumulation than plants treated with ABA on leaves, but not as much prolin e accumulation as in WT plants under

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45 abiotic stress. These results suggest that H2O2 may be able to stimulate ABAinduced proline accumulation. As noted by Verslues et al., (2007), the plant defense hormone salicylic acid (SA) can also regulate H2O2 levels and may interact with ABA (Guan and Scandali os, 1995). All of these data together suggest a highly complex form of signa l-transduced reaction mechanisms and potential interplay with photorespirati on. From this study, small pieces of the system have been laid on the table, leaving an enticing puzzle of ABA-proline accumulation mechanisms. Follow-up studies focused on measur ing possible buildup of glyoxylate or H2O2 in the peroxisome may enlighten us with a more comprehensive understanding of exactly how these mutant s were functioning differently than a WT GGAT protein. There was a clear increase in H2O2, but the paper did not discuss which process within the plant cell was creating the increased levels of H2O2. From the evidence accumulated, one c ould conjecture that their mutants may have a somewhat decreased catalytic ac tivity due to the fact that the previous enzymatic reaction (the transformation of glycolate into glyoxylate via GLO) produces H2O2 (Foyer et al., 2009). Slower catalytic activity of GGAT might produce a buildup of glyoxylate and H2O2 2.3.1.4 Glycine decarboxylase and seri ne hydroxymethyltransferase Glycine decarboxylase (GDC), found in mitochondria, is a multienzyme system composed of four different pr oteins: P, a 200 kDa homodimer; H, a 14 kDa monomeric lipoamide-containing prot ein; T, a 41 kDa monomeric protein

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46 requiring THF cofactor; and L, a 100 kDa homodimer containing FAD (Douce and Neuberger, 1999; Douce et al., 2001; Engel et al., 2007). The GDC catalyzes the following reaction (Douce et al., 2001): Eq. 2.1: 2 glycine + NAD + H2O serine + CO2 + NH3 + NADH + H+ In GDC’s catalytic reacti on, the H-protein plays a crucial role by ferrying the substrate between the other three pr oteins’ active sites. The substrate undergoes reductive methylamination in th e P-protein, methylamine transfer in the Tprotein, and electron transfer at the L-protein. All of the proteins that make up the GDC complex can be disassociated and behave as independent proteins (Douce et al., 2001). By acting as a complex, the H-,P-,L-, and Tenzymes catalyze oxidative carboxylation and deamination of glycine, producing NH3, CO2, and NADPH/NADH. The remaining carbon, the met hylene carbon of glycine is used in the so-called one carbon (C1) metabolism (Engel et al., 2007). The one carbon methylene is transferred to 5,6,7,8-tetrahydrof olate (THF), which is then used to form N5, N10 –methylene5,6,7,8tetra hydropteroylpolyfolate (CH2 –THF). CH2 –THF reacts with a second molecule of gl ycine to form serine via the enzyme serine hydroxymethyltransferase (SHMT). The rate of CO2 release from the GDC can produce as much as five times that of the citric acid cycle (Krebs/T CA cycle) which is also found in the mitochondria. Furthermore, the rate of glycine metabolism by single leaf

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47 mitochondria in a C3 plant can exceed 1200 nmol of glycine converted to serine per minute. To accomplish this rapid ra te of glycine conversion, the GDC and SHMT are found in exceptionally high nu mbers in the mitochondria, to such an extent that they comprise about half the proteins in spinach and pea leaf mitochondria (Oliver, D.J. et al., 1990; Douce et al., 2001). At these levels (0.3 g/ml in the mitochondria), GDC and SHMT distinctly alter the density of the mitochondria organelles (Vauclare, P. et al., 1996; Douce et al., 2001). GDC contributes to bo th photorespiratory and C1 metabolism in all biosynthetically active tissues Reactions involving the C1 pathway are essential to all organisms, and in plants supply the units needed to synthesize proteins, nucleic acids, pantothenate (vitamin B5), and a great variety of methylated molecules (Hanson and Roje, 2000). Accord ing to a current hypothesis on plant C1 metabolism, serine acts as a vehicle to transport CH2 from the mitochondria to other compartments in the cell, mainly to the cytosol, where CH2 –THF is resynthesized by SHMT isoforms to feed a multitude of biosynthetic mechanisms (Engel et al., 2007). The remaining glycine is rec onverted into serine by GDC and mitochondrial SHMT to be reused in this glycine-serine cycle. It has been postulated that the glycine-serine cycle is necessary for C1 metabolism (Mouillon et al., 1999; Engel et al., 2007). Engel et al., (2007) wanted to test Mouillon et al. ’s (1999) hypothesis. They created mutant strains of GDC, and studied how the mutation affected plant health. To produce the GDC mutants, they isolated 2 T-DNA insertion lines for the two P-protein genes found in Arabidopsis thaliana AtGLDP1 and AtGLDP2.

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48 In the individual knockout lin es, the plants displayed no difference in appearance in growth than the WT while grown in the same standard conditions. Western blotting displayed no clear reduction of th e P-protein content from both individual knockout lines, suggesting that the rema ining Pgene was overexpressed to compensate for the loss of the missing Pgene. However, when they produced a GDC mutant with a double knockout of both Pprotein genes, the plants were affected very badly. Even when grown in a high CO2 environment with moderate light intensity, the plants that were able to survive were very small, whitish in color, and experienced a growth arrest at the cotyledon stage. When the scientists tried to keep the double knockout GDC plants alive, growing them in a high CO2 environment with low light intensity, th ey did not survive for longer than 3-4 weeks (Engel et al., 2007). This suggests that GDC activity is necessary for plant function beyond the photorespiratory cycle and stress protection. From studies on C1 metabolism, it has been suggested that serine synthesis can occur by using CH2 –THF synthesized from formate via the C1 –THF synthase system. This would provide a secondary route for the C1 metabolism (Li et al., 2003). To ensure that this hypothesized system could not allow the pl ants a modest survival, they added different concentrations of 0.1 to 2 mMol formate to the medium, but that did not coax any more vitality out of the plants. Due to the GDC double mutant’s comple te inability to survive in a hostile environment or otherwise, it could be s uggested that the photorespiratory pathway is vital for the success of a plant. GDC ma y play a vital role in processes other

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49 than photorespiration, such as perhaps the C1 pathway. The results also suggest that the mitochondrial conversion of glyc ine to serine (als o executed by another photorespiratory enzyme, SHMT), represents a requisite component of C1 metabolism in all plant tissues (Mouillon et al., 1999; Engel et al., 2007). 2.3.1.5 Hydroxypyruvate reductase Hydroxypyruvate reductase (HPR) is a photorespiratory enzyme responsible for catalyzing the reaction of hydroxypyruvate to glycerate with the assistance of the energetic molecule NADH (Mano et al., 1997). Earlier on, it was stated that deletion of any one of the core photorespiratory enzymes would cause severe sensitivity to mutants grown in normal atmospheric environments. The enzyme HPR may be the exception to this rule (Timm et al., 2008). HPR transcripts have been demonstrated to be both developmentally regulated and enhanced by light (Mano et al., 1997). Another interesting feature about HPR is that, unlike most other photor espiratory enzymes, this enzyme is located in both the plant peroxisome and the cytosol. Of the two HPR enzymes, HPR1 has a carboxyl-terminal tripeptide of se rine, lysine, and leucine, which is a well known targeting signal to microbodi es. HPR2 lacks this carboxy-terminus, suggesting that it remains in th e cytosol of the plant cell (Mano et al., 1997). HPR’s uniqueness of location in the pe roxisome and the cytosol, as well as the HPR mutant’s survival in photores piratory conditions may be related. In a study by Murray et al., (1989) they isolated a barl ey mutant that displayed severely reduced activity of HPR1. Desp ite the fact that one of the main

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50 photorespiratory enzymes was severely depressed, the mutants were able to maintain 75% of CO2 fixation rates as compared to WT. However, later research by Timm et al., (2008) describes a slightly different story. In Timm et al.’s work, they identified HPR2, the version of the HPR enzyme found in the cytosol. By developing mutants with HPR1 absent, HPR2 absent, and both HPR1 and HPR2 absent, they were better able to study the significance of these enzymes. The HP R1 and HPR2 single mutants had a phenotype that was very similar to the WT. However, the double mutant strain was found to display the characteristic photorespiratory mutant phenotype. When testing the levels of hydr oxypyruvate found in the mutant s, they found a nine-fold increase in the double knockouts and a sixfold increase in the peroxisomal HPR1 mutants. Even with the HPR2 mutants, they noticed a slightly higher (but not statistically significant) level of hydroxypyruvate (Timm et al., 2008). HPR1 and 2 were capable of surv iving in the normal atmospheric environment, but displayed reduced growth even in comparison to the single HPR knockout lines. Characteristic to photor espiratory mutants, once the HPR double knockouts were housed in an enriched CO2 environment, the mutants were then able to display a phenotype like WT plan ts grown in the normal atmospheric environment (Timm et al., 2008). The HPR double mutant’s phenotype in normal atmospheric conditions suggests that both enzymes in the peroxisome and the cytosol might play a part in photorespiration with evidence pointing towa rds HPR1 acting as the main enzyme and HPR2 acting as an overload system. This so-called ‘cytosolic bypass’ is not

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51 yet fully understood, but it ha s been suggested that th ese enzymes’ dual function might be dynamically regulated by the avai lability of NADH in the context of peroxisomal redox homeostasis (Timm et al., 2008). 2.3.1.6 Glycerate kinase Glycerate kinase (GLYK), found in th e plant chloroplast, catalyzes the final reaction in the photorespiratory C2 cycle by reacting with glycerate to produce 3PGA, an intermediate in the Calvin Cycle (Husic et al., 1987; Boldt et al ., 2005). Despite its importance to th e photorespiratory pathway, GLYK’s primary structure and encoding genes have been only somwehat recently uncovered (Boldt et al., 2005). According to research done by Boldt et al., (2005) one gene, AtGLYK, appears to encode GLYK in Arabidopsis thaliana With this information in mind, the team produced a GLYK knockout to as sess the affects this might have on the plant. When culturing the knockouts in normal atmospheric conditions, Boldt et al., (2005) unsurprisingly found that thei r mutants displayed the signature photorespiratory phenotype found in mo st other mutants. In an ambient atmosphere, the plants were able to surv ive for up to two weeks but their growth became arrested in the early cotyledon stage. Once housed in a CO2 enriched environment, the plants were fully viab le and fertile, but continued to display reduced growth as compared to the WT.

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52 Furthermore, Boldt et al., (2005) found up to a 200-fold accumulation of glycerate in the mutants, as well as a five-fold increase in the upstream metabolites hydroxypyruvate and serine. Thes e data demonstrate that GLYK is most likely the only enzyme capable of converting glycerate into 3PGA. The mutant phenotype points once more toward s the necessity of photorespiration for plant survival. 2.3.2 Nitrogen uptake/reuptake Nitrogen, an element necessary for the production of amino acids and proteins, plays a major role in plant gr owth and development. Despite being the most abundant element in the atmosphere (N2), nitrogen is often the limiting nutrient for plants in most natural environments. In orde r to combat this issue, plants have come up with varying mechan isms to acquire, assimilate, and recycle nitrogen (Bernard and Habash, 2009). Ge netic and biochemical studies have indicated that the GS/GOGAT cycle is the primary pathway for NH assimilation (Suzuki and Knaff, 2005), and nitrogen r eassimilation is the rate-limiting step in photorespiration (Wallsgrove et al., 1987; Hausler et al., 1994; Kozaki and Takeba, 1996). Additionally, since glycine oxidation via GDC in the mitochondria can release 10 to 20-fold amounts more nitr ogen than through primary assimilation, it is clearly an im portant step in photorespiration (Potel et al., 2009). Glutamine synthetase (GS) and gl utamate synthase (GOGAT) together represent a major mechanism for C3 angiosperms to assimilate and recycle NH3

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53 produced from the GDC complex into us eful biological molecules (Miflin et al., 1980; Weber and Flugge, 2002). GS, with the assistance of ATP, catalyzes the fixation of NH3 to the -carboxyl group of glutamate to form glutamine (Bernard and Habash, 2009). On the other hand GOGAT, a.k.a., glutam ine: 2-oxoglutarate amidotransferase, catalyzes the reductan t-driven transfer of the amide amino group of glutamine to 2-oxoglutarate to yi eld two molecules of glutamate (Forde and Leas, 2007). 2-oxoglutarate, the subs trate necessary for the GOGAT enzyme conversion, is synthesized in both the mitochondria and the cytosol. The 2oxoglutarate is then transported into the chloroplast via a 2oxoglutarate/malate translocator (DiT1) (Webber and Flu gge, 2002). Glutamate, the product of the reaction with GOGAT, can also be transfe rred by a glutamate/malate translocator (DiT2) out of the chloroplast and into the peroxisome for use in the GGAT photorespiratory reaction. These transl ocator molecules link cytosolic and plastidic nitrogen assimilation, and are e ssential for plant metabolism (Weber and Flugge, 2002). Figure 2.2 : The GS-GOGAT cycle, located in the chloroplast. Ammonia produced in the mitochondria is converted into glutamine by glutamine synthetase (GS) and ATP. Glutamate synthase (GOG AT) next reacts with glutamine and 2oxoglutarate to form two molecules of glutamate (Bernard and Habash, 2009).

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54 2.3.2.1 Glutamine synthetase Glutamine synthetase (GS), found in the chloroplast and the cytosol, has been described in three different forms. GSI and GSII are found in both prokaryotes and eukaryotes, though proka ryotes tend to have more GSI and eukaryotes tend to have more GSII (Ber nard and Habash, 2009). The third version of GS, GSIII, has been found in bacteri odes, butyvibrio (a bacterium that produces fermentation products of acetat e, lactate, butyrate, formate, H2 and CO2) (Fuller, 2004), and some cy anobacteria (Garcia-Dominguez et al., 1997; Bernard and Habash, 2009). Estimates on the evolution of the GSI and GSII enzymes suggest the genes arose from genetic duplication some time around 3500 Mya, making it possibly one of the oldest exis ting and functioning genes (Bernard and Habash, 2009). To give a frame of referen ce, the schism between plant and animal kingdoms is thought to have occurred 1200 Mya (Bernard and Habash, 2009). Chloroplastic GSII, found to be 44-45 kD a in size, is the most abundant in higher plants although GSI is still found in some species of higher plants (Oliveira et al., 2002). In every higher plant studied so far, GSII is coded by a single gene, while GSI is coded by up to 5 different genes depending on the plant species (Oliveira et al., 2002; Broyart et al., 2010). GSII also tends to be found more often in leaf mesophyll cells, while GSI is much more common in vascular cells, specifically phloem (Oliveira et al., 2002). Because of these differences, it is thought that GSI and GSII serve distinctly different purposes (Lea and Forde, 1994; Oliveira et al., 2002). Cytoplasmic GSI, which is slightly smaller than GSII

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55 at 38-40 kDa, has not been well studied and its function is not yet elucidated, although it seems to be involve d in the assimilation of NH3 from the soil and the metabolic recycling of NH3 from other pathways th an photorespiration (Kirby et al., 2006). It has been proposed that sin ce GSI is expressed in the vascular bundles, it might play a role in the trans port of glutamine into other plant organs (Carvalho et al., 1992; Kirby et al., 2006; Broyart et al., 2010). Additionally, work by Fuentes et al., (2001) and Oliveira et al., (2002) suggest GSI may play a role in the regulation of photosynt hesis (as reviewed in Fuentes et al., 2001; Oliveira et al., 2002; Forde and Leas, 2007; Bernard and Habash, 2009). The atomic structure of maize cytosolic GS has been uncovered, indicating that the enzyme forms a decamer ic structure composed of two face-toface pentameric rings of subunits, with a total of 10 active sites formed between every two adjacent subunits in the pentamer (Unno et al., 2006; Bernard and Habash, 2009). In a study by Kozaki and Takeba, (1996) transgenic tobacco lines were created that were reduced or enriched in plastidic GSII. When comparing the metabolic activities of transgenic ove rexpressed GSII line 10, the underexpressed GSII line 21, and control plant SR1, they came to many interesting conclusions. When comparing the three lin es with a wild-type contro l burst in photorespiration that occurs in the dark directly after transfer from light, the control plants displayed rapidly decreased photores piration. The overexp ressed GSII line however continued to photorespire fo r a much longer time, while in the underexpressed GSII line photorespiration wa s very low and diminished very

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56 rapidly. This clearly establis hes the role of GSII as a key photorespiratory enzyme in nitrogen recycling. Kozaki and Takeba (1996) next studi ed the changes in ETR (electron transport chain) of the three lines du ring photosynthesis und er high light in CO2free air. In normal atmospheric conditions and normal light, the ETRs for the high GSII strain, control, and low GSII st rain were respectively 118, 105, and 50 Mol electrons m-2 s-1 in the plants. Initial ETR was high in all lines, indicating that photorespiration can drive el ectron transport in the ab sence of photosynthesis. After the high illumination, ETR dropped for all plants, but the decrease in the increased GSII line was smaller than that of both other lines. Furthermore, the ETR of the underexpressed GSII plants fe ll to almost no ETR activity after 1 hour, indicating photoinhibition. Sequentia lly, the mildest to hardest hit by inhibition was the increased GSII line, th e control line, and the decreased GSII line. This result suggests that the level of activity of GSII in th e leaves of a plant correlates with their capacity of photorespiration and tolerance of higher-intensity light, and thus photoinhibition. In another study by Oliveira et al., (2002), mutants of Nicotiana tabacum were produced by inserting cytosolic GSI gene or chloroplastic GSII from a pea plant ( Pisum sativum ) under the control of a cau liflower mosaic virus (CMV) promoter. Interestingly enough, all transg enic lines overexpressing GSII displayed a cosuppressed phenotype with no expr ession of GSII mRNA and drastically reduced levels of both GSI and GSII pr oteins. Furthermore, they displayed chlorosis, reductions in the levels of fr esh weight, dry weight and soluble protein.

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57 When grown in high CO2 or supplemented with glutamine, the chlorotic phenotype disappeared. Due to the cosuppr ession, these GSII mutants resemble GSII-deficient photorespiratory mu tants of barley (Wallsgrove et al., 1987; Lea and Forde 1994; Oliveira et al., 2002), which can also survive with the supplementation of glutamine and a high CO2 environment. These results indicate that the overand underexpressing transgen ic GSII plants die from depletion of amino donors from the pool of organic nitrogen, caused by their inability to reassimilate photorespiratory NH3 (Oliveira et al., 2002). Transgenic lines for their GSI transgenic plants consistently displayed the highest level of GS activity, as well as the highest le vels of fresh weight, dry weight, and soluble protein compared to the control plants (Oliveira, et al., 2002). The GSI-overexpressing transg enics displayed increases in fresh weight in both nitrogen limiting and non-nitrogen limiti ng conditions, and performed best under conditions of moderate light and high inorganic nitrogen. Even with no extra nitrogen, the GSI-overexpressing transgen ic plants still outgrew the control plants. This suggests that the transgen ic GSI plants might have a growth advantage due to a more efficient pro cessing and recycling of photorespiratory NH3. In further studies on the GSI plants w ith respect to light, it was found that there is a direct correlation between incr eased levels of cytosolic GSI in the transgenic plants and increased le vels of photorespiration (Oliveira et al., 2002). However despite these increased levels of photorespiration, the mutants were still outproducing the control plants in several different area s: serine/glycine ratios

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58 displayed a 3.5-fold increase, there was a two-fold increase in glutamate levels, and a 6.3 to 7-fold reduction of the total levels of free NH3 in the plant. This was because despite the increase in photorespiration, the CO2 compensation point (the point at which CO2 consumption by photosynthesis equals the rate of CO2 evolution by photorespiration from th e GDC) remained unchanged (Oliveira et al., 2002). Thus, it can be inferred that phot osynthesis must have increased along with increasing photorespiration, ma king GSI a possible regulator of photosynthesis (Oliveira et al., 2002). 2.3.2.2 Glutamate synthase In plants, glutamate synthase (GOGAT ) occurs in two of three distinct forms. The first form (Fd-GOGAT) tends to be present in photosynthetic tissue and uses reduced ferredoxin as the elect ron donor. The second form of GOGAT uses NADH as its electron donor, and is more predominantly found in nonphotosynthetic tissue (Forde and Leas, 2007) Both forms of these enzymes are found in the chloroplast or other plastid s (Suzuki and Knaff, 2005). The third form, NADPH-GOGAT, is found in bacteria (Reitzer, 1996; Su zuki and Knaff, 2005). For photosynthetic tissu e, the enzyme can utilize light energy as a source of reductant. On the other hand, nonphotosynthetic tissue obtains NADH from the pentose phosphate pathway (Forde and Leas, 2007). In a recent study by Potel et al., (2009) the team identified the genes coding for Fd-GOGAT (GLU1 and GL U2) and NADH-GOGAT (GLT) in Arabidopsis thaliana They found that the GLU1 knockout mutant was reduced to

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59 less than 3% GOGAT activity as compar ed to WT, while the GLU2 knockout nearly achieved WT levels in both norm al atmospheric conditions and elevated CO2. These data suggest that the GLU1 gene codes for the major Fd-GOGAT enzyme. Additionally, GLT activity, which accounts for 3% of the total GOGAT activity in WT plants, was reduced to a quarter of the activity in the GLT knockout mutant (Potel et al., 2009). The GLU1 mutant was found to ha ve accumulated a large amount of photorespiratory NH3 after having been transferred from a high CO2 environment to the atmospheric environment two days earlier. The GLT mu tant had slightly increased photorespiratory and nonphotorespiratory NH3, while the GLU2 knockout displayed WT levels of NH3. In a high CO2 environment, GLU2 and GLT both displayed reduced glutamate levels and increased glutamine levels, demonstrating that they primarily f unction in nonphotorespiratory nitrogen assimilation (Potel et al., 2009). In another study by Ishizaki et al. (2009) the author s created mutant GLU1-overexpressing lines in order to study the changes in the amino acid pools in varying conditions (i.e.high light, CO2 concentration, and nitrate concentration). Only one of their line s, 18-4, had drastic overexpression with a 62% increase. The other two lines, 13-1 a nd Z20-5, showed slight but statistically significant increases in gl utamate generation rate. Under high light intensity (300 mol-2 m s-1), two of the three GLU1 mutant lines experienced more than a 10% increase in as partate, threonine, lysine, histidine, and leucine than the WT. Th e amino acid increase was highest for

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60 aspartate, with a maximum of 57% increase in production. Despite these differences in amino acids, there was no phenotypic difference between the mutants and WT. In a high CO2 environment, all mutant GLU1-overexpressing lines displayed increased levels of thes e amino acids in comparison to WT with the exception of phenylalanine. In a variet y of different conditions (transitioning from moderate to strong light in normal and high CO2, and low and high levels of nitrogen paired with low and high levels of light intensity ), they found some differences with the WT. The level of glut amate remained more or less stable in the overexpressors as compared to the WT, while aspartate and threonine increased significantly under most conditions for the mutants. All of these data suggest that the increased expression of the photorespiratory enzyme GOGAT might in fact lend the transgenic plant stress tolerance. 2.3.3 H2O2 metabolism 2.3.3.1 Catalase Catalase is perhaps the longest studied enzyme ever, with the first biological characterization reported in 1900 (Loewen et al., 2000). Catalase, which is found in the plant peroxiso me, catalyzes the reaction (Loewen et al., 2000): Eq. 2.2 : 2 H2O2 2 H2O + O2 Due to the prevalence of catalase in both plants and animals, the enzyme was relatively easy to isolate. Catalase structure and sequence vary from one organism to the other, with a broad range of subunit sizes, a variety of quaternary

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61 structures, at least two different pros thetic groups, and substantially differing sequences (Loewen, et al., 2000). There are three different groups of catalase enzymes: typical/monofunctional catalases found in Eubacteria, Archaeabacteria, Protista, Fungi, Plantae, and Animalia; catalase-peroxidases found in all of the previously stated Kingdoms except Pl antae and Animalia; and manganese catalase, with a dimanganese active site, found in a minor bacterial protein family. For the most part, monofunctional or typica l catalase enzymes display only minor peroxidase activity, unlike the catalase-per oxidases, and can be further sorted on the basis of subunit size. The first two gr oups of catalases, the typical catalases and catalase-peroxidases, are heme enzymes (Zamocky et al., 2008). The reaction of catalase with H2O2 occurs in two distinct stages, as stated in equations 2.3 and 2.4. (Loewen et al., 2000): Eq. 2.3 : Heme-FeIII + H2O2 Heme-FeV =O (Cpd 1) + H2O Eq. 2.4 : Heme-FeV =O + H2O2 Heme-FeIII + H2O + O2 In the first step, the heme-iron complex is oxidized to form compound 1 (Cpd 1), which is an oxyferryl species w ith charge delocalized on the heme. The second step involves the reduction of ne wly formed compound 1 using a second molecule of H2O2 as the electron donor. The two st eps of catalase reactions occur at a very fast speed, with a general turnover rate of >105 molecules/second (Loewen et al., 2000). In plants, catala se scavenges H2O2 generated during mitochondrial ETC, oxidation of the fatty acids, and most of all in the peroxisome during

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62 photorespiration. Accumulating evidence sugge sts that catalase plays a major role in plant defense, aging, and senescence (Loewen et al., 2000). Most higher plants contain three different sets of catalase genes. In a study by Queval et al. (2007) the expression of one of the catalase genes (named CAT2) was disabled. As a beginning to their study, they grew mutants and WT plants in normal atmospheric conditions in standard irradiance for Arabidopsis thaliana (200 mol quanta m-2 sec-1), which accounted for about 50% saturation of photosynthesis. Under these growing c onditions, the CAT2 mutants displayed severely decreased growth in comparis on to the WT. As expected, when the CAT2 mutants were grown in an increased CO2 environment, there was no difference between mutant and wild type phenotype, suggesting that CAT2 has a function specific to the photorespirato ry cycle. When studying the levels of corresponding transgenics in WT Arabidopsis thaliana they found that CAT 2 and CAT 3 were the most highly expresse d genes, while CAT 1 transcripts were much less abundant. Despite the loss of function of CAT2 in the CAT mutants, there was no sign of compensatory induc tion of CAT1 in either atmospheric condition. Transfer of WT plants and mutants to ambient air from a high CO2 environment did not cause a change in the number of catalase transcripts. Despite this fact, catalase activities were induced twice as strongly (Queval et al., 2007). This suggests that the separate CAT genes are not functionally redundant, and may be expressed in distinct ways. Despite the fact that the mutant CAT2 plants displayed decreased growth under normal atmospheric conditions, no lesion formation occurred (which often

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63 occurs from decreased CAT2 activity, as was found in other studies such as Dat et al., 2003). However, when the CAT2 mutants were grown at the same irradiance for longer periods of time (12 and 16 hours respectively), the plants displayed extensive lesions, with plants under the 16 hour sunlight regime the most badly affected. When testing the CAT2 muta nts’ levels of redox cofactors and antioxidants in comparison to the WT, th ey found that the greatest oxidation in the mutant’s glutathione pool. Both NAD/NADP and ascorbate pools were similarly expressed in th e CAT2 mutant and WT. Glutathione is a chemical that serves multiple functions in the plant metabolism. While glutathione is most not ably found to play an antioxidant role the chloroplast, it has also been demonstrat ed to play, to a lesser extent, the same roles in the mitochondria, cyto sol, and peroxisome (Noctor et al., 2002). Changes in the status of glutathione have been implicated to affect multiple biochemical reactions, such as glutathionylation reac tions, calcium signaling, and activation of transcription factors (Queval et al., 2009). Queval et al. (2007) found a clear trend between time spent in sunlight and a decrease in total glutathione (GSH). This decrease in glutathione was also accompanied by a decrease in the disulfid e form of glutathione (GSSG) in longer periods of sunlight as well. An analysis of H2O2 marker transcripts exhibited that upregulation of CAT2 depended on daylight in a similar way to glutathione: CAT2 was markedly induced in short da ys, but was less strongly upregulated as daylength increased. Altoge ther, this data suggests that photorespiratory H2O2dependent gene expression may be influenced by photoperiod.

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64 2.4 Conclusions In this chapter, a far more rich a nd detailed story of the photorespiratory reactions was outlined. The source of the previously noted phenomenon on stress tolerance were given a more thorough i nvestigation, and general ideas about the usefulness of photorespiration were fleshe d out into more s ubstantive evidence. While the in-depth workings of phot orespiration are now clarified, the mechanisms by which they are regulated have not yet been summarized in this chapter. Already it has become apparent that photorespiration appears to play an important role in stress resistance and nitrogen assimilation. However certain experiments as those conducted by Oliveira et al., (2002) highlight the fact that the photorespiratory mechanism might be ha rnessed for other beneficial uses as well, namely in the effort to engineer a more productive plant. Chapter 3 will go into more detail on the scientific community’s efforts to produce a plant with greater CO2 assimilation and photosynthetic efficiency.

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65Chapter 3. Efforts to improve photosynthetic efficiency 3.1 Introduction As discussed in chapter 1, there is much inefficiency linked to the photorespiratory reactions. Due to the fact that Rubisco evolved in a high carbon dioxide and low oxygen environment, the ove rall mechanism and ‘design’ of this unique enzyme did not initially take th e low specificity for carbon dioxide as opposed to oxygen into account. Because of this inefficiency, there have been many scientific efforts to either reduce or remove the photorespirato ry process altogether. Several different strategies have been utilized in the quest for increased efficiency (and thus the goal of increased biomass of plants ) by reducing photorespiratory activity. Because the many plant biochemical pro cesses are so deeply interconnected, manipulation of one portion or system of the photosynthesizing mechanism can affect the rest. For the photorespiratory pathway, the most direct way of reducing or eliminating photorespiration is via experimentation with Rubisco, Rubisco activase, and by converting C3 into C4 plants. However, is the reduction of photores piration a practical way to produce a more productive plant? As will be discu ssed, research and manipulation of the Calvin cycle, photosystems, and the photor espiratory pathway (fig. 3.1) do not necessarily equate to a reduction of photorespiration, but may be a key to producing a more successful plant.

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66 Figure 3.1 : A diagram of the different system s and proteins that have been modified in the effort to improve the efficiency of photosynthesis. The system shown in green is photosynthetic electron transport chain (PETC), the system in blue is the Calvin cycle of photos ynthetic carbon reductive chain (PCRC), the system in red is the photorespirative or C2 cycle, the protein in black is the enzyme Rubisco, and the protein in purple is Rubisco activase (RCA). Not shown in this picture are the efforts to convert C3 plants into C4 plants, and the use of nanomolecules to increase effi ciency of photosynthesis (Gao et al., 2006; Gao et al., 2008. With exception of nanomolecules, re ferences reviewed in Peterhansel, Neissen, and Kebeish, 2008). Chapter 3 will outline a general overview of some of the many strategies attempted in an effort to increase orga nic carbon in the biosphere by manipulating the systems illustrated in figure 3.1. This chapter is divided into two sections: research devoted to reducing C2 metabolic activity, and efforts to increase the rate of photosynthetic activity. By manipulating these different systems using a variety of techniques, it is thought by some th at the quest to produce a more productive plant may one day be achieved.

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67 3.2 Efforts to reduce the C2 pathway 3.2.1 Rubisco Due to its ability to c onvert inorganic carbon diox ide into organic carbon, the holoenzyme Rubisco is vital to life on earth (Andersson and Backlund, 2008). Every year, an estimated 4 x 108 metric tons of carbon di oxide are conv erted into organic carbon via the process of phot osynthesis (Kannapan and Gready, 2008), 45% of which is produced from mari ne phytoplankton (Andersson and Backlund, 2008). Yet despite Rubisco’s essential role in life on earth, this enzyme is far from efficient. Compared to other hi ghly productive enzymes which can catalyze reactions at speeds of up to 108 molecules s-1, Rubisco runs at a snail’s pace of 3.3x 10 s-1 (Spreitzer, 1993). Also, as noted in chapter 1, Rubisco has a lack of specificity for its subs trate of atmospheric CO2 and will also react with O2 (Spreitzer and Salvucci 2002; Andersson and Taylor, 2003). What makes this holoenzyme particularly inefficient, however, is not just its low specificity and catalytic turnov er, but the additional hindrance of the oxygenation reaction’s products. The photorespi ratory reactions needed to convert 2-phosphoglycolate back into 3PGA expend en ergy carriers and result in a loss of between 30-50% of the carbon fixed (Rai nes, 2006; Kannapan and Gready, 2008). This lack of catalytic efficiency ha s implications for crop yield, nitrogen utilization and water consumption, as well as implications related to global temperature (Andersson, 2008). Because of its many perceived flaws, Rubisco is

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68 naturally a prime candidate for genetic engineering with th e hopes of creating a more efficient molecule. The push to produce a more catalytically favorable enzyme has resulted in many different strategies and techniques w ith varying degrees of success. Most experiments aiming to produce a more useful enzyme focus on studying or altering the following elements of or relate d to Rubisco: residues found in or near the active site, residues important to stru cture (especially secondary structure, as noted in Andersson 2008), the residu es important for activation of the holoenzyme, the protein (Rubisco activase ) needed to activate the holoenzyme, and the chaperones and chaperonins needed to properly fold Rubisco. By studying the nature of these elements of Rubisc o, it is hoped that science might one day elucidate a highly effective enzyme. The parameters for producing an effective Rubisco enzyme are the following: a Kcat/ Kc quotient of at least 108 molecules CO2/ second (where Kcat is CO2–saturated carboxylase activity), a higher Kc than the CO2 concentration in the chloroplast stroma (which is commonly 8 M), and perfect specificity (Sc/o) for CO2 relative to O2 (Andrews and Whitney, 2003). Currently, the Rubisco molecule has a Kcat/ Kc somewhere between 8.2 x 104 to 3.2 x 105 (from tobacco and Rhodospirillum rubrum respectively), a Kc between 9.3-10.7, and Sc/o between 12-82 (Andrews a nd Whitney, 2003). Clearly, the effort to design a perfect Rubisco enzyme is an upward battle. 3.2.1.1 Site-directed mutagenesis

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69 Site-directed mutagenesis is a system of changing a specific residue, in this case in the hope of producing a mo re efficient enzyme. Site directed mutations can be focused on a number of different parts of the enzyme, and can be used to introduce one or more residue changes. For example, site directed mutagenesis could be used to alter impor tant residues in the active site, residues near the active site, or re sidues pertinent for the over all enzyme conformation. In the case of Rubisco, site-directed studies focusing on altering residues in or near the active site have mostly produced catal ytically compromised versions of the protein (Parikh et al., 2006). For example, a study by Du et al., (2003) and Spreitzer et al., (2005) focused on the study of transforming the Rubisco of Chlamydomonas reinhardtii an aquatic green algae. In both studies, they attempted to convert the catalytic properties of C. reinhardtii ’s Rubisco to that of a terre strial plant’s Rubisco. The hope for this research is to garner an unde rstanding of the structural basis of the differences between the two different forms of Rubisco in an effort to produce a more efficient Rubisco. The terrestrial fo rm of Rubisco has a higher specificity than that of C. reinhardtii so it would hypothetically aid the algae if its specificity could be increased. Du et al., (2003) used site-directed mutagenesis to convert Met-42 and Cys-53 of C. reinhardtii to land plant Val-42 and Ala-53 to see if they could increase the algae’s specificity for CO2 in the large subunit N-terminal domain. When little change was observed between mutant and WT, they further experimented by changing C. reinhardtii methy-Cys-256, Lys-258, and Ile-265 at the bottom of the / –barrel active site into land plant residues Phe-256, Arg-258,

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70 and Val-265. With these changes, the mu tant line suffered a 10% decrease in CO2 specificity, largely due to an 85% decreas e in carboxylation catalytic efficiency (Du et al., 2003). Work by Spreitzer et al., (2005) continued the work done by Du et al., (2003). Along with the previously stat ed site-directed mutations of the C. reinhardtii Rubisco, the authors decided to change residues between the -strands A and B of the small subunit, which are know n to effect catalysis. Site directed mutagenesis of C. reinhardtii ’s Val-221 and Val-235 residues to land plant Cys221 and Ile-235 caused the CO2 specificity to return to terrestrial plant levels. Additional substitutions to the AB loop caused a 12-17% increase in specifity. Despite this, the enhanced CO2/O2 specificity was less than that of terrestrial plants. However, the enhanced CO2/O2 specificity did not make for a better enzyme, seeing as the decreased Kc and Vc resulted in a reduction of catalytic efficiency (Spreitzer et al., 2005). Additionally, there has been accumulating evidence pointing towards the idea that Rubisco may already be fu lly optimized through the process of evolution. This idea is evinced by the inverse relationship between Rubisco’s specificity versus its catalytic efficienc y, typically seen when comparing Rubisco enzymes across species (Long et al., 2006; Peterhansel et al., 2008; Andersson, 2008). Depending on fluctuating environmental conditions, one of these two values becomes more important for plant success (Peterhansel et al., 2008). For example, during drought, specificity becomes more vital than catalytic rate due to stomatal closing, which limits photosynthesi s due to a lack of gas exchange. On

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71 the other hand, during high light conditions it becomes more favorable for the plant to have reduced specificity and a greater catalytic rate (Peterhansel et al., 2008). The inverse relationship of specificity versus catalytic activity is not an absolute rule, seeing as some Rubisco enzymes from red algae do display high specificity values at decent catalytic rates (Peterhansel et al., 2008). This fact has helped scientists hold out hope on the id ea that Rubisco is not yet perfectly evolved to its environment, which leads us to the next section. 3.2.1.2 The concept of directed protein evolution The concept of directed protein evol ution is the attempt to evolve an enzyme towards a specific purpose or optim al performance. By generating a large mutant library and then scr eening them for traits speci fic to the purpose, progress towards producing a more functional enzy me might be made (Mueller-Cajar and Whitney, 2008). With this strategy, there are two ways a more efficient Rubisco enzyme can be achieved: by either rando mly evolving a more efficient protein by speeding up evolution in th e laboratory, or by disti nguishing important residues by isolating the genes that code for the Rubisco “overand u nderachievers”. In order to speed up the process of evolu tion, the Rubisco gene in question is horizontally transferred into an or ganism with a high turnover rate. Two different screens have been used to obtain relevant information on Rubisco: finding the mutants which di splay a highly productive phenotype, and finding the mutants which display a highly unproductive phenotype (Mueller-

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72 Cajar and Whitney, 2008; Andersson a nd Backlund, 2008). This process of evolving mutants hinges on finding or ge netically engineering a photosynthetic organism with a high transformation rate a nd a rapid life cycle in order to make a large mutant library (Mue ller-Cajar and Whitney, 2008). The unicellular green algae Chlamydomonus reinhardtii has been of particular usefulness in screening for phe notypic mutant “underachievers”. Due to the fact that C. reinhardtii possesses the form I Rubisco found in most higher plants and can survive without photosynthe sis via the supplementation of acetate, Rubisco mutants which possess very poor or completely ineffective photosynthesizing capabilities can still survive and be identified for study (Mueller-Cajar and Whitney, 2008; Andersson and Backlund, 2008). 3.2.1.3 The use of horizontal transfer for potential plant improvement As noted by Peterhansel et al., (2008), the tendency of there to be an inverse relationship between catalytic efficiency and specificity is not absolute. The thinking is that the greater specific ity Rubisco has for carbon dioxide and the greater the catalytic ra te, the lower the chance the plant has to undergo photorespiration and the more productive th e plant might be. Thus, scientists came up with the idea of producing a more productive plant via horizontal transfer. Horizontal transfer is defined to be the process in which an organism incorporates genetic material from anot her organism through a means other than reproduction. In the context of plant productivity and reduced photorespiration,

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73 there are many ways to advantageously us e this technique: on e can find a Rubisco enzyme with better cataly tic activity and specificity than those found in crop plants and transfer the genes coding for the Rubisco enzyme into the crop plant, or can isolate the gene for a Rubisco enzyme they want to optimize, such as Synechococcus PCC6301 and place it in an organi sm with a rapid life cycle (in this case E. coli ), as done by Parikh et al., (2006) to promote rapid evolution. However, researchers have had many st umbling blocks while trying to explore these areas of research. The first issue lies in the fact that Rubisco is a very large enzyme which needs the assistance of many different pr oteins for both assembly and regulation of activity. Up until this year, obtaining an in vitro reconstitution of the active form of the Rubisco from its unfolded subunits in high yiel d was not possible (Ellis, 2010). A study by Liu et al. (2010) finally made the breakthrough for proper folding of the Rubisco enzyme, giving up to a 40% yield of functional enzyme (Liu et al., 2010). It would be particularly advantageous to horizonta lly transfer higher plant Rubisco genes into high thr oughput organisms like bacteria however this exercise has not yet been successfu lly accomplished due to improper folding of the enzyme (Peterhansel et al., 2008). After an uphill battle several years ago, the reverse experiment of transf erring bacterial Rubisco into higher plants has been accomplished, as evidenced by a study done by Whitney and Andrews, (2001). The most feasible pathway of this study was to insert red algae Rubisco genes (which are more favorable due to the f act that they do not follow the inverse

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74 relationship of catalytic e fficiency vs. specificity) in to the genome of a higher crop plant. In most higher plants and gr een algae the genes coding for the Rubisco subunits are divided, the large subunit’s (RbcL) genetic information in the plastome and the small subunit’s (RbcS) genetic information in the nucleus (Whitney and Andrews, 2001). In red algae, both RbcL and RbcS are housed in an operon in the chromosomes and plasmi ds of prokaryotes and plastomes of nongreen algae (Whitney and Andrews, 2001). In this study by Whitney and Andrews (2001), the authors used plasmid transformation to replac e Rubisco in tobacco ( N. tabacum ) with a simple homodimeric form from the -protobacterium Rhodospirullum rubrum because it requires no small subunits or special assembly. Despite the fact that R. rubrum ’s Rubisco enzyme does not seem to fo llow the inverse re lationship between specificity and catalytic activity the transg enic tobacco lines, unfortunately, had to be supplemented with CO2 (Whitney and Andrews, 2001). 3.2.2 Efforts to improve Rubisco activase As discussed earlier in chapter 1, the activation state of Rubisco is regulated by Rubisco activase (RCA). RCA can remove sugar phosphate inhibitors from active and inactive (carbamylated and decarbamylated) Rubisco via protein-protein interac tions and ATP hydrolysis, t hus regulating the enzyme (Kurek et al., 2007; Kumar et al., 2009). While Rubisco is a thermostable enzyme that displays increased catalytic turnover at temperatures up to 45 C, RCA has been found to be extremely

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75 thermolabile. RCA has been demonstrat ed to being the rate-limiting enzyme in vitro due to denaturation, leading to reduced photosynthesis (Kurek et al., 2007; Mueller-Cajar and Whitney, 2008). Because of this instability, production of a more stable RCA could prove to be particularly useful in the attempt to produce a more productive plant. With this knowledge in mind, Kurek et al., (2007) used a technique known as gene shuffling in the attemp t to produce a more thermostable A. thaliana RCA. In the process of gene shuffling, a fam ily of related genes are chopped up with enzymes, and the resulting gene fragment s are heated up to separate them into single-stranded templates. Some of these fragments will bind to other fragments that share complimentary DNA regions. Th e regions that are not complimentary hang over the ends of the templates. Th e PCR reaction treats the complimentary regions as primers, and new double helical DNA is built. However, PCR also adds bases to the overhanging piece of the pr imer, forming a new double helix there as well. This creates a chimer ical DNA structure (Cohen, 2001). The authors first developed a high throughput screen for thermostable variants of A. thaliana RCA that directly assayed Rubisco via gene shuffling. Further studies of these lines found that the three lead mutants produced in the screen coded for either a single mutation (Thr-274 to Arg-274), a triple mutation (Phe-168 to Leu-168, Val-257 to Ile-257, Lys-301 to Asn-301), or a quadruple mutation (Thr-274 to Arg-274, Phe-168 to Leu-168, Val-257 to Ile-257, Lys-301 to Asn-301). Under controlled growth conditions, the mutated RCA enzymes

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76 significantly improved CO2 assimilation, growth, and s eed yield under periods of moderate heat stress (Kurek et al., 2007; Mueller-Cajar and Whitney, 2008). In another study by Kumar et al., (2009), the authors took a slightly different tack in their attempt to pr oduce a more thermotolerant RCA. They created a chimeric hybrid RCA which took elements from N. tabacum and A. thaliana The RCA chimera was mostly based on the tobacco activase, but had the A. thaliana Rubisco recognition domain (Kumar et al., 2009). The RCA of N. tabacum is more thermotolerant than that of A. thaliana, so they wanted to test to see if they could confer greater thermostability to the plant A. thaliana by producing a mutant line with this chim eric RCA. Indeed, the chimera RCA was able to retain the thermostability of the tobacco activase, while still being able to activate the A. thaliana Rubisco. Root and vegetative growth was improved at all stages examined, and the mutants displa yed better seed and germination rates (Kumar et al., 2009). 3.2.3 Efforts to convert C3 plants into C4 plants If one wants to reduce or eliminate activity of the C2 pathway in plants, a good place to start would be by studying orga nisms that have already successfully reduced or eliminated the photorespirato ry reactions. Thus, researchers turned towards C4 plants in their quest to find the holy grail of plan t productivity (Holy hand grenades of Antioch excluded). As discussed earlier, the C4 plant metabolism evolved a biochemical pump, or carbon concentrating mech anism (CCM) that increases the

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77 concentration of CO2 (Ci) near Rubisco, strongly suppressing the rate of oxygenation and effectively re ducing the flux of the C2 cycle (Peterhansel et al., 2008). C4 photosynthesis has independently evol ved upwards of fifty times, 24 of which are attributed to the grass and sedge family (Roalsom, 2009, as reviewed in Sage and Sage, 2009). This metabol ism requires both physiological and anatomical adaptations: in all but two lineages ( Bienertia and Sueada genera), evolution of the C4 pathway is associated with the specialized Kranz anatomy (Sage and Sage, 2009). As discussed in chapter 1, the Kranz anatomy is an evolutionary modifica tion of the leaf into a mesophyll where phosphoenolpyruvate (PEP) carboxylation occurs, and a bundle sheath compartment where Rubisco and th e Calvin cycle are housed. The CO2 captured in the mesophyll undergoes a series of reactions before being diffused in the form of a C4 acid into the bundle sheath cells, where a molecule of CO2 is later released from the acid (Peterhansel et al., 2008; Sage and Sage, 2009). From here, the primary CO2 acceptor (PEP) is regenerated at the expense of ATP (Taniguchi et al., 2008). In the two plant lineages where the C4 metabolism displays no Kranz anatomy, C4 photosynthesis occurs all in one ce ll, though Rubisco is localized in a different region of the cell than PEP carboxylase (PEPC) (Sage and Sage, 2009). Additionally, some aquatic plant diat oms and higher plants also undergo C4 photosynthesis in only one cell, though they do not display segregation of carboxylation and decarboxylation steps (Sage and Sage, 2009).

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78 In all of the 7,500 terrestria l plants found to use the C4 metabolism, all but three express the Kranz anatomy, and all productive C4 crops have Kranz anatomy (Sage, 2004). This suggests that in order to produce a more efficient C4 plant from a C3 plant, the Kranz anatomy might also need to be transferred, which would undoubtedly require th e alteration and introducti on of several genes. Indeed, the genes and developmental pr ocesses needed to express the Kranz anatomy are largely unknown, which suggest that engineering the Kranz anatomy into C3 plants might be a particularly di fficult endeavor (Sage and Sage, 2009). Despite these issues, the existence of C4 photosynthesis occurring in one cell raises the possibility of engineering a C4 plant from a C3 plant. Likewise, if researchers can find a way to express Kranz anatomy in C3 plants, the possibility of converting a C3 plant into a C4 plant with reduced photorespiration may be possible (Sage and Sage, 2009). In a study by Taniguchi et al., (2008), four C4 enzymes (a maize C4– specific PEPC, orthophosphate dikinase (PPDK), a sorghum NADP-malate dehydogenase (MDH), and a rice C3–specific NADP-malic enzyme (ME)) were all expressed in the C3 rice plant. With these enzymes, they hoped to introduce the C4–like pathway of Hydrilla verticillata into the mesophyll cells of rice. They decided to attempt to add the C4 pathway of Hydrilla because it is a facultative C4 plant that shifts from C3 to C4 photosynthesis under low CO2 conditions without any need of specialized anatomy. In Hydrilla, upon the shift to C4 photosynthesis, the genes expressing C4 enzymes are upregulated. A ltogether, they found that overproduction of all four C4 enzymes very slightly improved photosynthesis in a

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79 few of the lines of transgenic rice, but at the same time caused a concomitant slight stunting of th e plants (Taniguchi et al., 2008). In order to run this experiment, five different gene constructs were used: the intact maize gene for PEPC and PPDK, the full-length cDNA for overproduction of NADP-ME, the rice ME isoform, and the sorghum MDH. These constructs were cloned into a binary vector containing a hygromycin resistance gene and introduced into rice plants via Agrobacterium tumefaciens – mediated gene transfer. Plants with th e highest protein expression were assumed to be homozygous and then selfed, and tran sgenic plants overproducing more than one enzyme were produced by crossing two different single transformants and/or by introducing a second gene into transformants (Taniguchi et al., 2008). Tanaguchi et al., 2008 found that overproduction of any single C4 protein had either no affect or reduced assimilation of CO2. In leaves of C3 plants, it has been found that PEPC has an anaplerotic (a reaction that restores a depleted metabolic intermediate) role in replen ishing the tricarboxylic acid cycle (TCA) with intermediates, which are then wit hdrawn to produce amino acids via nitrogen assimilation/reassimilation (a process linked with photorespiration). Agreeing with this information, overproduction of PEPC has been found to enhance respiration of rice leaves exposed to li ght. However because of this, these same plants displayed a slight re duction in assimilation of CO2 (Fukayama et al., 2003, per Taniguchi et al., 2008). Taniguchi et al .’s plants also displayed this inefficiency; however it took over a 50-fol d increase of PEPC to induce stunting of the transgenic plants. In a different study, Ku et al., (2000) found that

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80 overexpression of PEPC enha nced stomatal opening in rice plants and thus increased CO2 assimilation. Taniguchi et al .’s plants did not e xpress this benefit of enhanced stomatal opening. Overproducti on of PEPC did incr ease the rate of dark respiration, but this still did not substantia lly affect photosynthesis. Out of the many combinations of C4 enzymes being overexpressed, only overproduction of all four enzymes had positive affects on photosynthesis, and these positive effects were limited. The reduction of CO2 assimilation from overexpressed PEPC was recovered by overe xpression of all three other enzymes, and in a few cases modestly increased CO2 assimilation. Due to the fact that transgenic plants overexpressing PEPC PPDK, and ME did not display any increased CO2 assimilation, Taniguchi assumed that MDH activity must be necessary for the restoration or enhancement of photosynthesis. ME overproduction alone has been found to raise the NADPH/NADP+ ratio in the stroma, and thereby incr easing the chances of photoinhibition (NADP+ is needed to accept excited electrons in the photosystem) (Taniguchi et al., 2008). In most forms of C3 photosynthesis, you are getting either positive or no net gain of NADPH; in the case of NADP+–malic enzyme photosynthesis, 1 molecule of NADPH is produced for every 1 molecule of C4 acid that is being decarboxylated (Buchanan et al. 2000). Altogether, it is unknown whether or not the C4 pathway even functions in these quadruple transgenic plants. Even if the C4 pathway is functioning, it does not seem to create a CCM like it does in proper C4 plants.

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81 3.3 Efforts to increase metabolic rate While Rubisco and RCA make a ttractive candidates for genetic experimentation, there are many other ave nues of study worth pursuing that might uncover the secret to a more productive pl ant. The process of photosynthesis is far more complicated than these two important enzymes, so a broader view of this particular challenge is necessary. In order for photosynthesis to be accom plished, several systems have to be working in concert with each other: th e photosystems must provide energetic molecules, the Calvin cycle must use the energetic molecules to produce sugar, and the C2 pathway must recycle the produc ts from the Rubisco oxygenation reaction. In total, the flux of thes e metabolites throughout these pathways influences the overall production of hexose suga rs, and thus are an integral part of the goal to produce a more efficient plant. Recent studies with antisense RNA t echnology have allowed us to reduce the expression of metabolic enzymes and structures in these various pathways, allowing us to identify rate-limiting steps in photosynthesis (Rodriguez et al., 2007). By increasing expression of thes e rate-limiting genes, the goal of producing a more efficient plant may come one step closer to being a reality. 3.3.1 The Calvin cycle The Calvin cycle plays a central ro le in plant metabolism by providing intermediates for starch, succinate bi osynthesis, isoprenoid metabolism, and shikimic acid biosynthesis (Lefebvre et al., 2005). Due to its key role in

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82 photosynthesis, experimentation with the Calvin cycle could prove to be very useful in the endeavor to pr oduce a more efficient plant. Because of the vast importance of th e Calvin cycle, studies were done to measure the contribution that individual enzymes exert over the flux of the whole Calvin cycle metabolism. A wealth of knowledge was obtained from these studies. For example, it was found that in an environment with moderate light and temperature, more than 50% of WT levels of Rubisco could be removed before an effect on photosynthesis was observed. Ho wever under conditions of high light, reduction of Rubisco directly correlated to reduction of photosynthesis (Lefebvre et al., 2005). Through similar studies which reduced expression of a gene coding for particular enzymes, it was found that the enzymes G3P dehydrogenase, fructose1,6-bisphophatase (FBPase), phosphori bulokinase (PRKase) and plastidial aldolase were also highly overexpressed in the wild type (meaning that there was no reduction in photosynthesis until at le ast a 50% decrease in expression of the genes) (Lefebvre et al., 2005). On the other hand, even a small reduction in the number of transcripts of transketolase (TK) and sedoheptulose-1,7-bisphosphatase (SBPase) affected overall photos ynthesis in pl ants (Miyagawa et al., 2001; Lefebvre et al., 2005). TK and SBPase both hold unique and important functions in the Calvin cycle. Transketolase catalyzes three revers ible reactions in the regenerative phase of the Calvin cycle: the conversion of GAP to ribose 5-phosphate, the conversion of sedoheptulose 7-phosphate into xylulo se 5-phosphate, and the conversion of

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83 xylulose 5-phosphate back into GAP (Buchanan et al., 2000). A 20-30% reduction of TK resulted in a decline of carbon fixation and a decrease in phenylpropanoid metabolism (Lefebvre et al., 2005). SBPase catalyzes the irreversible reaction of se doheptulose 1,7bisphosphate into sedoheptulose 7-phosphate with release of Pi (Buchanan et al., 2000). SBPase functions as the branch poi nt between regeneration of RuBP and export to starch biosynthesis. It was found that when SBPase expression was reduced, RuBP regenerative capacity (or Jmax) also declined at a linear rate. Additionally, total biomass was reduced in antisense SBPase plants (Harrison et al., 2001; Lefebvre et al., 2005). The data on TK and SBPase suggests th at these enzymes might mark ratelimiting steps in the Calvin cycle reacti ons. Thus because of this information, experiments were conducted that incr eased the expression of both enzymes. Lefebvre et al., (2007) produced a transgenic line that had increased TK content via expression of an Arabidopsis thaliana TK cDNA. The transgenic plants displayed severe bleaching in the inte rveinal mesophyll cells, though the areas adjacent to these cells remain green. Lefebvre et al., (2007) suggested that this phenotype was the result of perturbed flow of carbon in the plastid, which is supported by data analysis of the metabolites in the plants (Lefebvre, et al. 2007). Studies with SBPase proved more fruitful. Lefebvre et al., (2005) used a full-length SBPase cDNA to prepare a sens e gene construct in the binary vector. The SBPase gene was inserted into Arabidopsis thaliana via the CMV 35s promoter, and the resulting plants were crossbred for four generations. The

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84 SBPase expression varied in the mutants: the range of SBPase expression ranged from close to WT levels, to up to 150% increased expression of SBPase (Lefebvre et al., 2005). Lefebvre et al. (2005) found that the operati ng efficiency of PSII was higher in plants with increased SB Pase expression (0.495-0.504 0.004, as compared to WT 0.460 0.003). Nonphotochemical quenching (NPQ) in the transgenics was not significan tly different than in WT pl ants, suggesting that this increase in photosystem activity might be used to support higher rates of photosynthetic activity. Studies with gas exchange found that the photosynthetic rate in young transgenic plants far ou tstripped young WT plants; however, this disparity receded when the plants grew to full maturity, and little difference in photosynthesis between the transgenics a nd WT were detected. There was an observed 12% increase in carbon fixed da ily in young plants, and only a 6% increase in carbon fixed daily in mature plants (Lefebvre et al., 2005). Lefebvre et al. (2005) also found a correlation between increased SBPase activity and carbohydrate accumulation. Plants with higher SBPase activity accumulated up to 50% more sucrose and starch than the WT. Even though photosynthetic activity of the mature plants was not increased, levels of sucrose and starch were still higher in muta nts most highly expressing SBPase. The SBPase mutants displayed higher rates of photosynthetic carbon fixation than WT plants at Ci (intercellular CO2) levels between 150 mol mol-1 to 1,200 mol mol1 (CO2 saturation). In fact, there was a posit ive correlation of SBPase activity and assimilation rate at both normal atmos pheric concentrations and saturated

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85 concentrations of CO2 (Lefebvre et al., 2005). In concordance with their previous conclusion, they found that the average area and weight of leaves in the early to mid-vegetative phase was 50% greater th an the WT, while in later stages of development there was little to no difference between the mutants and the WT (Lefebvre et al., 2005). Altogether, Lefebvre et al. found that light-saturated rates of photosynthesis measured under bot h prevailing and saturated CO2 environments increased linearly in response to SBPase activity. However strangely enough, this increase in photosynthesis itself was not in proportion to tota l SBPase activity. Lefebvre et al suggested that regulat ory properties of the system might result in the Calvin cycle being stunted. For example, they noted that SBPase activity is regulated by its product as well as thiodorexin (Raines et al., 1999; Lefebvre et al., 2005). Interestingly enough, an ear lier experiment which also targeted SBPase proved more fruitful. Miyagawa et al. (2001) designed an experiment where they produced transgenic tobacco plants to express the cyanobacterial FBPase/SBPase gene. In their study, they produced fifteen antibiotic-re sistant tobacco transformations via Agrobacterium tumefaciens -mediated transfer and then bred them. The total SBPase activity derived from plastidic SBPase (from the tobacco plant) and cyanobacterial SBPase/FBP ase increased by 2.3 0.4 and 1.7 0.1 in transformant lines TpFS-3 and TpFS-6 as compared to WT. Additionally, the total FBPase activity derived from plastidic FBPase as compared to cyanobacterial FBPase/SBPase displayed increases of 1.8 0.2 and 1.3 0.1 in the same two

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86 lines as compared to WT (Miyagawa et al., 2001). Unlike Lefebvre et al., 2005’s plants, Miyagawa et al. ’s (2001) transgenic plants continued to experience increased photosynthetic capacity and growth into full maturity. Miyagawa, et al.’s (2001) plants grew significan tly faster and larger than the WT. After 18 weeks of growth, the transgenic TpFS-3 and TpFS-6 plants displayed a 1.4 and 1.5-fold increase in heig ht as compared to the WT, with an equivalent increase in dry weight. Despit e these increases in growth, there was no evidence to suggest that to tal protein content per squa re meter increased in the transgenic plants. Nonetheless, the ac tivity of Rubisco in TpFS-3 and TpFS-6 lines was 1.2 and 1.1-fold higher than th at of the WT, indicating an increased activation of Rubisco. The overall photosynt hetic activity of the transgenic lines was also respectively 1.24 and 1.20-fold hi gher than the WT, and the levels of RuBP were 1.5 and 1.8-fold higher than that of the WT as well. Other metabolites (PGA, DHAP, F6P, G6P) were found to be at least 1.2-fold higher in the transgenic plants as compared to the WT, and Calvin cycle metabolism products (sucrose, starch, hexose) were at least 1.6-fold higher in the transgenic plants as compared to WT. One point of interest in this st udy was figuring out why the rate of photosynthesis under atmospheric CO2 increased in the transgenic plants. After referring to Farquhar et al., (1980), Miyagawa et al., (2001) suggested that the increase in RuBP might result in the higher activity of RCA, which could account for the 1.1 to 1.2-fold increased activity of Rubisco in the transgenic plants (although the level of activation may not account for the total effect).

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87 Further studies on increased expres sion of SBPase also displayed interesting finds. Feng et al., (2007) cloned a full-length rice SBPase cDNA, and introduced the plasmid into rice via Argrobacterium tumefaciens The resulting transgenic plants were selfed three times, and the T3 progeny was used in a study to see what effects would resu lt with high temperature (Feng et al., 2007). It was found that at about 35 C, the growth ra te started to decrease in WT, control (transformed, but with the SBP sequence omitted), and transgenic plants. However, the decrease in growth rate wa s much more marked in control and WT plants vs. the transgenic pl ants at high temperatures, particularly around 40-45 C. Coinciding with these numbers, the CO2 assimilation rate of all plants started to decrease significantly at 35 C, howev er the WT experienced a much sharper decrease in assimilation than the transgen ic plants. Additionally, when the plants were allowed a 2 hour period of recovery in 25 C following a 2 hour period in 45 temperatures, the transgenic plants fared far better than the WT. One of the transgenic lines, L5, was able to make a complete recovery of CO2 assimilation, while the WT plants were only able to make a 20% recovery of CO2 assimilation (Feng et al., 2007). Continuing on this str eam of thought, Feng et al., (2007) found that the content of RCA in soluble fractions decrea sed at high temperatures, and that this decrease was significantly larger in WT plants than transgenic plants. The changes in the content of RCA were largely reversible in transgenic plants, while the WT only showed a small recovery. Furthermore, the content of RCA in

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88 thylakoid fractions increase d at high temperatures, though this increase was more significant in WT as compared to transgenic plants (Feng et al., 2007). Altogether, these results indicate th at high temperatures cause RCA to associate with the stromal side of the t hylakoids. This association seems to be much higher in WT plants than transgen ic plants. Somehow, increased activity of SBPase may stabilize RCA and prevent th e enzyme from interacting with the thylakoid membrane under heat stress. A dditionally, the increase in SBPase also seemed to enhance the phosphoribulokina se (PRKase) activation state at high temperatures as well. Feng et al., (2007) suggest that this might occur because the increased expression of SBPase provi des increased amounts of ribulose 5phosphate (Ru5P), the reactant of PRKase The increased amount of Ru5P might then enhance the PRKase act ivation state, thus increas ing thermotolerance of the transgenic plants (Feng et al., 2007). 3.3.2 The photosystems Antisense RNA experiments on the phot osystems have provided evidence that transgenic plants with decreases in the content of ferrodoxin or Rieske ironsulfur (Fe-S) protein from cytochrome B6f (Cyt B6f ) displayed correlating decreases in CO2 assimilation rates (Rodriguez et al., 2007). Armed with this information, Rodriguez et al., 2007 decided to investigat e the expression of FdNADPH reductase (FNR), a F AD-containing enzyme that catalyzes the final step in the PETC. Plants with a 20-70% reduc tion of FNR displayed growth arrest,

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89 chlorophyll degradati on, and impaired CO2 uptake. This marked FNR as a possible rate-limiting step in the PETC (Rodriguez et al., 2007). In order to further study the eff ects of FNR on the PETC, Rodriguez et al., (2007) produced transgenic t obacco plants expressing pea ( Pisum sativum ) FNR in their chloroplasts. Rodriguez et al .’s experiment produced up to a 6-fold increase in FNR, however the rate of photosynthesis did not increase. Instead, the transgenic plants displayed an increase in tolerance to photooxidative damage (Rodriguez et al., 2007). While this outcome is not quite what the researchers were aiming for, stress resistance is a ve ry important facet of plant metabolism and productivity that should not be ov erlooked. Production of a stress-resistant plant is of just as great importance as productivity in the coming years due to potential increasing extremes in climate due to global warming. In another experiment by Chida et al. (2007), these resear chers decided to turn their attention to a di fferent part of the PETC, namely plastocyanin (PC). Plastocyanin is carrier that accepts electrons from the Cyt B6f complex and ferries them over to PSI (Buchanan, et al., 2000). In algae, the heme protein cytochrome c6 (Cyt c6), which is thought to have been evolutionarily eliminated from land plants, fulfills this duty instead of PC (Chida et al., 2007). Although the structures of these two proteins are entirely diff erent, they both fulfill the same redox potential (Chida et al., 2007). Chida et al., (2007) introduced the Porphyra Cyt c6 gene back into the land plant Arabidopsis thaliana and selfed the plants. This produced a transgenic line that displayed a 1.2 -fold increase of Cyt c6 as compared to PC when

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90 compared in terms of molar ratio. They found 1.9 and 1.3-fold increases in transgenic plant vs. WT height 40 and 60 days after planting, respectively. The root lengths 40 and 60 days after planting were also significantly longer (1.4 and 1.3-fold increases) as comp ared to the WT. Chida et al., (2007) found that after 80-90 days of growth, there were no differe nces in root length between transgenic and WT plants. However, I wonder if they took into account the fact that they were growing their plants in pots with a limited area for growth. Nevertheless, Chida et al., 2007 found that the expression of Cyt c6 in A. thaliana promoted the growth of the plants in early stages. Chlorophyll content in th e transgenic plants enjoyed a 1.1 to 1.3-fold increase, along with increased levels of protein (1.1-fold increase), ATP (1.9 -fold increase), NADPH (1.4-fold increase), and starch (1.2 -fold increase). The CO2 assimilation capacity increased up to 1.862 0.304 in transgenic plants as compared to WT (Chida et al., 2007). Additionally, these differences were still observed in the plants 70-80 days after pl anting. I find this part icularly interesting, since one would think that these plants might have still enjoyed a concomitant increase in biomass production at these la te time points, bu t again, perhaps the plants became root-bound. Altogether, Chida et al.’s (2007) work seems to suggest that an enhancement of the PETC can be achiev ed by increasing the rate of electron transfer, even though it does not seem to be the rate-limiting step. Due to the fact that PC and Cyt c6 both have the same redox potentia l, I believe that the same

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91 increases in efficiency mi ght be obtained by increasi ng the expression of PC in crop plants. 3.3.3 The photorespiratory pathway Despite the fact that a good amount of research has gone into reducing or entirely eliminating the C2 pathway, there is still room to improve upon it in the efforts to produce a more efficient plant. Work by Kebeish et al., (2007) attempted to improve upon the C2 pathway not by eliminating it, but instead by modifying it. They introduced the E. coli C2 reaction mechanisms into A. thaliana chloroplasts (see figure 3.2) in an effo rt to reduce the loss of fixed carbon and nitrogen that occurs when 2PG is metabolized through A. thaliana’s normal C2 cycle. Many bacteria are able to metabolize glycolate as their sole source of carbon. The bacterial photorespiratory di ffers from the plant photorespirative pathway after 2-phosphoglycolate is converted by 2PG (2-phosphoglycolate phosphatase) into glycolate. Instead of continuing down the previously outlined sequence illustrated in Fig. 2.1, glycol ate is converted into glyoxylate by GDH (glycolate dehydrogenase), and glyoxyl ate is converted into tartronic semialdehyde (TSR) by GCL (gl yoxylate carboligase) (Kebeish et al., 2007). Kebeish et al., (2007) speculated that the addition of a bacterial C2 pathway into crop plants could improve e fficiency in several ways. Unlike higher plant C2 metabolism which releases CO2 in the mitochondria, the bacterial C2 pathway could release CO2 in the chloroplast, directly where it is needed. This

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92 could potentially increase the rate of CO2 assimilation. Because the final product of the bacterial C2 pathway is glycolate, ni trogen does not have to be reassimilated in an energy-requiri ng reaction. Finally, bacterial C2 pathway doesn’t require ATP to function, unlike the C4 pathway (Kebeish et al., 2007). Figure 3.2 : The plant and bacterial C2 pathways. The bacterial pathway is denoted in red, while the plant bacteria l pathway is in black. GDHglycolate dehydrogenase; GCLglyoxylate carboxylig ase; TSRtartronic semialdehyde reductase (Kebeish et al., 2007). The bacterial photorespiratory pathway was inserted into A. thaliana via step-wise nuclear transforma tion and post-translational ta rgeting of proteins to plastids. In total, three plasmids were us ed to insert the full metabolic pathway: one plastid encoding GDH subunits D and E, a plastid encoding GDH subunit F, and one final plastid enc oding GCL and TSR (Kebeish et al., 2007).

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93 Compared to WT, transgenic plan ts expressing GDH DE and F subunits (DEF), and GDH DEF, GCL, and TSR (GT-DEF) displayed significantly increased dry weights of roots and shoots. This increase was most marked in the GT-DEF plants. Additionally, the transgenic plants continued to display increased biomass even under environmentally stress ful conditions (i.e., high temperature, strong light). This makes sense, since it is under these stressful conditions when the C2 pathway is more heavily trafficked (Kebeish et al ., 2007). When measuring the post -illumination burst (PIB) as an indicator of CO2 release into the mitochondria via the plant C2 enzyme GDC, the transgenic DEF and GT-DEF displayed a ~ 30% reduction in activity as compared to the WT (Kebeish et al., 2007). On the other hand, CO2 release into the chloroplasts of both transgenic plants significantly increased as compared to WT. This is particularly interesting seeing as plant line DEF does not have GLC, the bacterial C2 chloroplastic enzyme needed to convert glyoxylate into CO2 and tartronic semialdehyde. This suggests that A. thaliana has an endogenous glycolateoxidizing enzyme in plant chloroplasts, an idea that was already suggested by Goyal and Tolbert (1996). The rate of CO2 assimilation under ambient conditions was clearly increased in GT-DEF lines, however this advantage disappeared in a CO2saturated environment. Due to the fact that the transgenic plants release CO2 into the chloroplast (which is also the pla ce where the Calvin cycle and Rubisco are housed), CO2 assimilation increased due to increased Ci in the chloroplast (Kebeish et al., 2007). While the positive affects of CO2 release have been

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94 questioned due to the chloroplast’s low re sistance of diffusion (Von Caemmerer, 2003), Kebeish et al., (2007) brings up several lines of data that suggest the release does in fact make a noticeable impact on CO2 assimilation. First, the PIB (which indicates CO2 release) was reduced in transg enic lines. If a release of CO2 in the chloroplast were no more useful than a release of CO2 in the mitochondria, then the PIB would have remained the same in the transgenic plants. Next, they found that oxygenation inhibi tion showed a strong negative correlation with the CO2/ O2 ratio in the vicinity of Rubisco in GT-DEF plants. Finally, they found that the CO2 compensation point (which provides an independent measure of the CO2/ O2 ratio in the chloroplast) suggests that augmentation of Ci is possible (Kebeish et al., 2007). Kebeish et al., (2007) argues that using the C2 bacterial pathway in higher plants is more effective than converting a C3 plant into a C4 plant due to the fact that the C4 metabolic pathway uses ATP. Additionally, the C2 bacterial pathway removes the need to reassimilate NH3, a process that requires energy, which might also explain why these transgenic plants fare so well (Kebeish et al., 2007). 3.4 Conclusions A veritable cornucopia of study has been dedicated to the engineering of a more productive crop plant. The researchers involved have utilized a wide variety of different tools and concepts in their effort to feed an ever-growing world. A key to the success of this ch allenge is to learn from the mistakes of previous researchers, and look for trends which might be of use.

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95 After having studied a great number of papers, it seems that attempts to reduce the C2 pathway have seen less success than attempts to increase the pace of multiple systems. However, there are other reasons that a reduction or elimination of photorespiration might be harmful to plan ts, which will be di scussed in the next chapter.

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96Chapter 4. Reactive oxygen species and their relation to photorespiration 4.1 Introduction Much like any other organism, plants respond to stressful environments. The outcomes of many abiotic stressors produce reactive oxygen species (ROS) in plants, which creates a condition known as oxidative stress. This condition has the potential to damage plant cells due to th eir reduced state. The majority of the ROS's harmful effects are due to their abi lity to initiate a variety of autoxidative chain reactions on fatty acids (Smirnoff, 2000; Ahmad et al., 2008), and extreme sensitivity of the thiol-modulated enzy mes has been documented since the 1970s (Foyer and Noctor, 2005). There are a num ber of different ROS that can be generated in the cell, such as OH-, O2 -, H2O2, H2O-, and singlet oxygen 1O2 (Slesak et al., 2007). Due to the fact that H2O2 is not a radica l or negatively charged molecule, it is much longer lived in the cell than any of the other ROS. This makes it much more likely to be used as a signaling molecule. It is thought that aquaporins might facil itate the movement of H2O2 as well as water between cells (Slesak et al., 2007). 4.2 ROS production in the plant cell The following stressors are capable of disrupting the balance of reduction of ROS vs. the quenching activity of antioxidants, which can produce oxidative damage: 1) high light intensity, 2) te mperature extremes, 3) drought, 4) high salinity, 5) herbicide treatmen t, 6) mineral deficiencies 7) air pollution, 8) heavy

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97 metals, 9) wounding, 10) UV light, and 11) pathogen infection (reviewed by Buchanan et al., 2000). The main sites of ROS production are the mitochondria, chloroplast, peroxisome, plasma membra ne and apoplast, as shown in figure 2.1 (Ahmad et al. 2008). A brief overview will outline where ROS are made in the plant cell. Figure 4.1 : A diagram of the evolution of ROS in different cellular structures as also described in the text. (Slesak et al., 2007). 4.2.1 The Chloroplast ROS is created in the chloroplast th ylakoid via activity from the PETC. Due to the fact that reaction centers PSII and PSI are responsible for reducing electrons, ROS production is sure to occur (Miller et al., 2009). The photoproduction of ROS by the PETC is la rgely modulated by climate. For example, water stress can produce an exce ss of energized electrons because of a lack of CO2 being fixed by Rubisco as a result of stomatal closure and a reduction

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98 of gas exchange. During this stress period, the excess of excited electrons produced by the PSI are transferred to oxygen to produce superoxide (O2 -1) via the Mehler reaction (fig 4.2) (Miller et al., 2009), while an ov erreduction at PSII produces singlet oxygen (Miller et al., 2009). Figure 4.2 : The Mehler reaction, or water-water cycle. In this diagram, reduced ferrodoxin reacts with oxygen to form super oxide radicals. This radical is then converted into another ROS, hydrogen peroxi de, with the assistan ce of superoxide dismutase. The cycle is finally finish ed when hydrogen peroxide and ascorbate react with ascorbate pero xidase to produce water. The O2 -1 is converted into H2O2 by a membrane-attached copper or zinc superoxide dismutase enzyme (SOD), and membrane-bound thylakoid ascorbate peroxidase (APX) reacts with ascorbic acid (vitamin C or AsA) and H2O2, converting them into water and monodehydroascorbate (MDHA) by the enzyme monodehydroascorbate reductase (MDHAR). From there, MDHA can be reduced

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99 back to AsA via ferredoxin. Thus, the reasoning behind the designation of this pathway as the ‘water-water’ cycle become s apparent: the process starts with the splitting of water in the PSII, and ends with the forma tion of water after reaction with APX (Heldt, 2005; Miller et al., 2009). The chemical equations of these reactions are as follows (Heldt, 2005): Eq. 4.1 : The Mehler reactionO2 + M(n-1) O2 + Mn+ Eq. 4.2 : Superoxide dismutase2 O2 + 2 H+ O2 + H2O2 Eq. 4.3 : Ascorbate peroxidaseH2O2 + 2 ascorbate 2 H2O + 2 monodehydroascorbate. H2O2 can also act as a reductant fo r metals, producing hydroxyl radicals (OH-). Hydroxyl radicals are hi ghly aggressive substances for which the plant has evolved no protective enzymes, and t hus the plant must deal with H2O2 immediately (Heldt, 2005). Dehydroascorbate (DHA) can be form ed if MDHAR does not reduce it, and DHA can be reduced back to AsA via dehydroascorbate reductase (DHAR) (Miller et al., 2009). DHAR links the Mehler reaction with the ascorbateglutathione cycle by oxid izing glutathione (GSH) and supplying DHA with the electron necessary to convert it back to being AsA (Buchanan et al., 2000). Additionally, thylakoid-associated peroxi redoxins (PrxR) and thioredoxins (Trx) can work together as another antioxidative mechanism, and act as an alternative to the Mehler reaction for H2O2 (Miller et al., 2009). The water-water cycle is consid ered the primary source of H2O2 in the chloroplast, and it is widely accepted as a vital alternative sink for reduced

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100 electrons (Slesak et al., 2007). Other superoxide re ductive pathways (which will be discussed later in more detail) do not contribute much to the addition of H2O2 in the chloroplast (Slesak et al., 2007). 4.2.2 The Peroxisome The oxidation of glycolate by the photorespiratory enzyme glycolate oxidase (GLO) takes up the lion’s share fo r production of ROS in the peroxisome (Noctor et al., 2002; Miller et al., 2009). However fatty acid -oxidation, the flavin oxidase pathway, xanthine oxidase, and dismutation of superoxide radicals also have a hand in peroxi some ROS evolution (Miller et al., 2009). The main antioxidative enzyme in the peroxisome is catalase, an enzyme linked with the photorespiratory cycle that converts H2O2 into water and oxygen. APX and the AsA-GSH cycle also contribute to H2O2 scavenging (Miller et al., 2009). 4.2.3 The Mitochondrion Though to a smaller extent than the chloroplast or the peroxisome, the mitochondrion also produces ROS (Slesak et al., 2007). The main sites of ROS production occur at the ETC’s NADP H dehydrogenases (complex I) and cytochrome bc1 complex (complex III) (Slesak et al., 2007). The superoxide formed in these areas are generally converted to H2O2 via a mitochondrionspecific Manganese SOD enzyme. While mitochondria are out-produced by chloroplasts and peroxisomes in the li ght, in dark or in non-photosynthesizing tissue, mitochondria can be a main source of ROS (Slesak et al., 2007). One

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101 reason the mitochondria might have a lower output of ROS is due to the fact that in plants, alternative oxidase (AOX) medi ates an alternative pathway to reduce oxygen, and thus competes with complex III for electrons (Slesak et al., 2007). Perturbation of the ETC has been show n to result in overreduction of the ubiquinone pool, and as such will produce ROS (Miller et al., 2009). 4.2.4 The Apoplast Recent studies have found that ROS production is vital for the apoplast. In Arabidopsis thaliana two genes coding for NADPH oxidases expressed in guard and mesophyll cells are responsible for pr oducing ROS that is needed to stimulate ABA (abscisic acid) –induced stomatal closure (Kwak et al., 2003; Slesak et al., 2007). Other ROS-forming enzymes ( cell wall associated oxidases and peroxidases, polyamine oxidases) are thought to be responsible for acclimation to stressful environments, and strengthening of the cell wall (Slesak et al., 2007). 4.2.5 The Cytosol In the cytoplasm, an ETC associated with the endoplasmic reticulum is the major source of ROS, where forms of cy tochrome P450 are involved in oxidation and hydroxylation reactions. Although the cy tosol is responsible for only a small portion of ROS, it may act as a sink for any H2O2 that leaks out of a plasmid or cellular structure (Slesak et al., 2007). 4.3 Photorespiration and ROS

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102 Photorespiration is implicated in the protection of plants from O2 by preventing the overreduction of the electr on transport chain (Kozaki and Takeba, 1996; Kebeish et al., 2007; Xu et al., 2009). If electron acceptors such as NADPH are not used (as what might occur if the stomata were to completely close), then the plant will have a shortage of NADP+ and will have to dissipate the excess energy via other pathways than photores piration, such as the Mehler and ascorbate reactions. These pathways, in turn, would be overloaded and incapable of neutralizing the damaging ROS (Kozaki and Takeba, 1996; Ahmad et al., 2008). To get a better understanding of electron flux in the plant cell, it is important to have a general grasp on the percentage of electron flow in these various pathways. The Mehler O2 reuptake reaction, which is the major sink for the chloroplast and peroxisome (as well as the greater producers of ROS in the cell), is estimated to support a flow of electron transport of only ~ 10% (Badger et al., 2000). This means that a majority of electron flow must be consumed by either the C2 or Calvin cycle. Considering the existence of significant levels of oxygenation to carboxylation in C3 plants, the C2 pathway almost assuredly assumes a vital role as an electron sink in these plants. Even with the highly reduced oxygenation reaction of C4 plants, one could still expect that the C2 pathway might play somewhat of a role as an electron sink. Additionally, downstr eam production of H2O2 may contribute to signaling pathways which help the plant manage st ress. A short overview of some signaling pathways will be discussed in the next section.

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103 4.4 ROS signaling of the MAPK kinase pathway While photorespiration may help prevent the formation of ROS by expending NADPH, it also in creases the amount of H2O2 found in the cell (Ahmad et al., 2008). The photorespiratory pathway produces H2O2 when glycolate reacts with O2 to form glyoxylate and H2O2 in the peroxisome (Buchanan et al., 2000). In recent years ROS such as H2O2 have been found to regulate a number of biological processe s, such as growth, the cell cycle, programmed cell death, hormone signaling, development, and biotic and abiotic stress responses (Mittler, 2004). Mittler, (2004) suggests that over the course of evolution, plants were able to achieve a high degree of control over ROS toxicity, and from that stringent control, are now capable of largely checking ROS toxicity while enabling ROS to act as signaling mo lecules. Control of ROS as signaling molecules seems to require a large netw ork of genes, as suggested by the 152 genes implicated in RO S regulation. (Mittler et al., 2004). In particular, the ROS H2O2 has been found to tr igger several mitogenactivated protein kinase cascades (MAPK) (Jonak et al., 2002). MAPK is a serine/threonine protein kina se that plays a leading role in the transduction of several other signals, both intr aand extracellular. In A. thaliana, there are 20 genes that encode putative MAPKs, 10 genes coding for MAPKKs, and 10 genes encoding for MAPKKKs (Hadiarto et al., 2006). The MAPK signaling pathways are known to regulate cell growth and d eath, differentiation, the cell cycle and stress responses (Jonak et al., 2002). The MAPK protein generally functions in a

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104 cascade where it is phosphorylated and activated by MAPK kinase (MAPKK), which is itself activated by MAPKK ki nase (MAPKKK). The MAP kinase signal transduction pathway is illustrated in figure 4.3. Figure 4.3 : A generic depiction of the mitogenactivated protein kinases (MAPK) signal transduction pa thway. Relevant to this di scussion, after signaling from hydrogen peroxide, the MAPK pathway is activated in a cascade. Receptormediated activation of MAPKs can o ccur through physical interaction or phosphorylation by the receptor (Ahmad et al., 2008).

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105 In Arabidopsis H2O2 specifically activates the MAPKs MAPK3 and MAPK6 via MAPKKK ANP1. Transgenic plants overexpressing ANP1 were found to have increased tolerance to h eat shock, freezing, and salt stress (Kovtum et al., 2000; Ahmad et al., 2008). H2O2 has also been found to increase the expression of Arabidopsis nucleotide disphosphate kinase 2 (NDPK2) (Moon et al., 2003; Ahmad, et al., 2008). NDPK2 is a protein known to regulate cell proliferation, development, and different iation. It has been suggested that the effect of NDPK2 may be mediated by MAPK3 and MAPK6 due to the fact that DNPK2 can interact and activ ate those specific MAPKs (Moon et al., 2003). In recent studies, a new signaling pa thway has been uncovered that plays an important role in leaf senescen ce. Leaf senescence is an intricate developmental phase that has been de monstrated to be involved in both degenerative and nutrient recycling processe s. It can be induced by a variety of abiotic stressors, such as wounding, poisoning, and increased levels of H2O2 (Zhou et al., 2009) Transgenic plants over-expressing the MAPKK9-MAPK6 pathway were found to senesce precociousl y, demonstrating that that this complex regulates leaf senescence (Zhou et al., 2009). Although somewhat counterintuitive, leaf sene scence is yet another protective mechanism for stressed plants: senescent leaves are a source of nutri ents for developing parts of the plants (Berberich et al., 1999). A rather recently isolat ed MAP kinase gene has been found to up-regulate its transcript level in response to salinity and cold stresses (Zhang et al., 2006; Ahmad et al., 2008). Additionally, several protein kinases including Arabidopsis

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106 thaliana MAPKKK1, are transcriptionally up-regulated under high salt conditions, as well as other abiotic stresses. Arabidopsis thaliana MAPKK1 (AtMKK1) has been found to have a role in abiotic stresse s such as wounding, cold, drought, and high salt stress by activating Arabidopsis thaliana MAPK4 (AtMPK4) (Jonak et al., 2002). Although details of the activation of the AtMKK1-AtMPK4 pathway has not been deciph ered, it is sensible to reason that this pathway might as well be mediated by ROS. Overall, these reports provide strong evidence that the H2O2-induced MAPK kinase signa ling cascades aid in the mediation of stress tolerance. 4.5 Conclusions: connections the C2 pathway and ROS If we take a step back to research done in chapter 2, we can make some definite connections between the phot orespiratory pathway and reactive oxygen species. For example, Queval et al., (2007) created mutants lacking the CAT2 enzyme, and found that plants grown in ambient conditions had severely decreased rosette biomass, intercellular redox pertur bation, and activation of oxidative signaling pathways. Additiona lly, they found that photoperiod was a critical determinant of stress response. While the C2 pathway was already used as a sink, perturbation of the levels of H2O2 in the peroxisome clearly caused severe issues in the CAT2 mutants. Hackenburg et al., (2009) studied a bacterial Synechoscystis sp. strain pathway in which 2PG (2 phosphoglycolate, the initial C2 metabolite) is converted by a bacterial glycine decarboxylase (Gcv). In this pathway, the Mehler reaction

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107 is mediated by flavoproteins which seem to work in concert with Gcv to dissipate ROS in the bacteria. In a study by Moreno et al., (2005), the authors found that SHMT recessive mutants (serine hydroxymethyltransferase, a C2 enzyme found in the mitochondria) were more susceptible to aberrant regulation of cell death. Since ROS signaling has already been connected to cell death, this suggests that SHMT plays a vital role in redox regulati on and a cell signaling pathway, farther downstream (Moreno et al., 2005). Though there are many connections with the C2 pathway and ROS, it is hard to quantify the contribution of the C2-generated ROS in plant signaling. The flow of electrons into different path ways is in continual flux, depending on species and the outside environment. Th e task of quantifying electron flow in each cellular structure is currently rather challenging, so there is no way to know exactly how important the C2 pathway is for stress response. Yet some researchers speculate that it does play a significan t role in redox regulation and signaling (Foyer et al., 2009). Even with our questionable unde rstanding of this particular dynamic however, there is re ason to believe that the C2 pathway has an important use. If we do accomplish the mission of e ngineering the photorespiratory pathway out of plants (which is a tall order), are we sure the plants will be able to endure environmental variation?

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108Chapter 5. Possible Future Initiatives 5.1 Introduction After an overview of photosynthesi s and photorespiration, discussion about photorespiratory mutants and thei r affects, a window into the current research strategies to produce a more effi cient plant, and a glimpse of the function of the C2 pathway, a humble understanding of this particular portion of plant metabolism can be reached. As more in formation is uncovered, even more questions and lines of study are opened, promising for an exciting future. The possibilities for research are endless; however there are certain lines of experimentation which, given the chan ce, may yield greater productivity. After having read and digested much of the studies focused on creating a more efficient plant, I took note of which experiments seemed to have the greatest success. Surprisingly very few successful experiments had anything to do with the remodeling of Rubisco, but instead increas ed performance in other interconnected photosynthetic systems. Of particular inte rest were studies pertaining to the C2 enzyme GSI, the Calvin cycle en zyme sedoheptulose 1,7-bisphosphatase, cytochrome C6 in the photosystems, and ventures into the engineering a bacterial C2 pathway in higher plants. All of these experiments were discussed in earlier chapters, so I will rehash only briefly the important elements of each of each article. 5.2 Metabolic Mutants

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109 In work done by Oliveira et al., (2002) they studied the affects of increasing expression of cytosolic GSI (glutamine synthetase), an enzyme in the nitrogen uptake/reuptake pathway conn ected to photorespiration (enzyme #10 from figure 2.1). Oliveira et al., (2002) found that their GSI mutants displayed a light-dependent improved growth phenot ype in both nitrogen limited and nonlimited conditions, as eviden ced by increases in fresh weight, dry weight, and soluble leaf protein (Oliveira et al., 2002). This study work achieved a rather significant success of producing a plant w ith improved output not by reducing or removing the C2 pathway, but instead by speed ing up the rate of reactions of most of the plant metabolic system. In another paper by Miyagawa et al., (2001), transgenic tobacco plants expressing a cyanobacterial gene en coding fructose 1,6/sedohepulose1,7bisphosphatase (FBPase/SBPase) displaye d increased photosynthetic activity and growth under normal atmospheric cond itions. Under irradiances below 1,500 mol/m2/s, the transgenic plants displayed a 1.2-fold increase in photosynthesis (Miyagawa et al., 2001). Similar to Oliveira et al.’s (2002) work, Miyagawa et al., (2001) were able to design a more productive plant not by decreasing photorespira tion or enhancing Rubisco catalytic efficiency, but instead by improving the rate of reaction in the Calvin cycle. Work done by Kebeish et al., (2007) shed light on another novel venture to design a more productive plant by cr eating a bacterial phot orespiratory bypass in a higher plant. The bacterial C2 system does not eliminate photorespiration

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110 occurring, but instead shunts it into a pathway which has less reaction steps, removes the necessity of reconvertin g nitrogen, and consumes less energy (Kebeish et al., 2007). Much like the other muta nts discussed so far, the transgenic bacterial C2 plants were found to grow faster, produce more shoot and root biomass, and produce more soluble s ugars. This clearly marks the mutants as more productive plants. Finally, work by Chida et al., (2007), is another example of a successful experiment where increased expression of an enzyme in the photosynthetic system produced more productive plants. In Chida et al.’s (2007) experiment, they produced transgenic Arabidopsis thaliana plants expressing the algal Porphyra Cytochrome C6 (Cyt C6) gene. Chida et al. found that the mutants expressing the algal Cyt C6 gene enhanced growth of the plants as characterized by height, leaves, and root growth. 5.3 Future Studies Enhancing plant productivity by in creasing the metabolic rate of photosynthesis displays great promise fo r future research. Due to its great potential for global agriculture, further st udy of the kinetics of the photosynthetic metabolism would be advantageous. While poring over the many studies be ing published, I thought of some experiments that might prove useful After having ta ken note of Chida et al., (2007), Oliveira et al., (2001), Kebeish et al., (2007), and Miyagawa et al.’s (2001) promising results, I propose the ge netic alteration of a species (such as

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111 Arabidopsis thaliana initially, and then agronomi cally relevant plants in subsequent studies) to create a series of transgenic plants which overexpress enzymes that have already been demonstr ated to increase plant efficiency and overall production via a cauliflower mosaic virus promoter constructs. I would like to see the production of a line of transgenics which overexpresses GSI, sedoheptulose 1,7-bisphosphatase/fructos e-1,6-bisphosphatase enzymes, Cyt c6, and the bacterial photorespiratory path way simultaneously. By increasing the number of transcripts for these genes, I would hope to increase the reaction pathway for nitrogen assim ilation, sugar production, and C2 activity (and reintroduction of CO2). In order to get a fuller picture of the genes being expressed, I would also like to see the creation of mu tants which singly express each of the aforementioned genes to be able to compare against the efficiency of the combinatorial mutants. Due to the nitrogen reuptake cycle’s connection with photorespiration, it might also be wise to prepare double mutants which express both the bacterial C2 pathway and the GSI gene to ask whether nitrogen reassimilation is affected the plants in question. The activitie s of each of the overexpressed enzymes would have to be measured, as well as gas exchange and chlorophyll measurements. The determination of metabolite and carbohydrate levels will have to be measured, as well as plant fresh weight and dry weight. Similarly, it may be beneficial to run experiments overexpressing plastocyanin, similar to what was done by Chida et al., (2007). With this information at hand, we can evaluate the benefits of non-terrestrial cytochrome c6 (Cyt c6) as compared to the terrestrial plasto cyanin (PC), and see if one of the two

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112 carrier proteins is more effective. A dditionally, a transgenic line overexpressing both Cyt c6 and PC might be of interest. By producing these transgenic lines we will gain a better knowledge of the metabolic flow of metabolites in plants while working towards producing a plant that is both a better producer and more stress tolerant. Instead of looking at the photorespiratory system as a burden that mu st be eliminated, it can be viewed as just another metabolic pathway that recycl es nitrogen, and plays a role in stress protection and possi bly cell signaling. 5.4 Conclusions With the promised of increased global temperature and increased population, the need to engineer a more produc tive crop plant is vi tal. In order to achieve this feat, one must look at the task at hand as an elaborate puzzle; in order to fit the puzzle pieces together, one mu st look at the task at hand from many different angles. After studying a modest selection of the ever-growing mass of information on this topic, it seems that the most re levant avenue of study is via increasing metabolic rate rather than reducing the C2 pathway. While attempts to reduce the C2 pathway have been clever, they have also become quite exhaustive. With information suggesting that photorespiration is more than just a wasteful pathway and in fact useful, it might be time for the scientific community to look at the challenge of producing more productive plants from a different angle.

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113 Appendix: Abbreviations 2PG2-phosphoglycolate 3PGA3-phosphoglycerate AAA+An ATPase family associated with diverse cel lular activities ABAabscisic acid ANP1A MAPKKK that activates MAPK 3 and 6 AOXalternative oxidase APXascorbate peroxidase AsAAscorbic acid AtGLYKGLYK gene AtMKK1/4Arabidopsis thaliana MAPKK1/4 AtPGLP1PGLP photorespiratory gene AtPGLP2Gene that codes for the n onphotorespiratory PGLP found in cytosol C1One carbon metabolism C2Photorespiratory cycle C3Calvin cycle C4The four carbon pathway CAMCrassulacean acid metabolism CATcatalase CAT1/2A CAT gene. CCMcarbon concentrating mechanism CH2-THF5,6,7,8-tetrahydropt eroyl polyglutamate CiThe concentration of CO2 CKABP2-carboxy 3-ketoarabitinol 1.5 bisphosphate CMVCauliflower mosaic virus Cyt c6heme protein cytochrome c6 from the photosystems DHADehydroascorbate DHAPdehydroxyacetone phosphate DHARdehydroascorbate reductase DiT12-oxoglutarate/ma late translocator DiT2glutamate/malate translocator ETCelectron transport chain ETRelectron transport rate FBPaseFructose 1,6-bisphosphatase Fd-GOGATA form of glutam ate synthase that uses fe rredoxin to obtain reduced electrons. FmThe maximum chlorophyll fluorescence value FMNflavin mononucleotide FNRFd-NADPH reductase F’q / F’mThe operating efficiency of PSII Fvthe maximum capacity for photochemical quenching Fv/Fmmax efficiency of photochemi stry after dark adaptation Fxferredoxin G3Pglycerate-3-phosphate GAPglyceraldehyde-3-phosphate

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114 GCLglyoxylate carboligase Gcvbacterial glycine decarboxylase gcvTA gene that codes for a T-protein in GDC GDCglycine decarboxylase GDHglycolate dehydrogenase GGATglutamate-glyoxylate aminotransferase GLOglycolate oxidase GLTNADH-GOGAT gene GLU1/2Fd-GOGAT genes GLYKGlycerate kinase GOGATGlutamate synthase GSGlutamine synthetase GSIChloroplastic GS GSIICytosolic GS GSI, GSII, GSIIIThe three different versions of glutamine synthetase GSHglutathione GSSGThe oxidized form of glutat hione; the disulfide form of GSH GT-DEFA transgenic line coding for GSH, GCL, and TSR HA 14 kDa monomeric lipoamide -containing prot ein from GDC H2Ageneric reductant HPRhydroxypyruvate reductase HPR1A form of HPR that has a carboxyl-ter minal tripeptide of serine, lysine, and leucine, which is a well known targeting signal to microbodies. HPR2A form of HPR that la cks the carboxy-terminus on HPR1 hv Light energy ICLisocitrate lyase JmaxRuBP regenerative capacity KcMichaelis-Menten constant for carboxylation Kcat/ KcKmMichaelis-Menten constant KoMichaelis-Menten constant for oxygenation LA 100 kDa homodimer c ontaining FAD from GDC LHCLight harvesting complex LOLow oxygen content MAPKmitogen-activated protein kinase MAPKKMAPK kinase MAPKKKMAPKK kinase MDHNADP-malate dehydogenase, a C4 enzyme from sorghum MDA/MDHAmonodehydroascorbate MDHARmonodehydroascorbate reductase MENADP-malic enzyme, specific to rice MLSmalate synthesis MyaMillion years ago NDPK2nucleotide diphosphate kinase NPQNonphotochemical quenchers odca gene that codes for oxalate decarboxylase

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115 PA 200 kDa homodimer from GDC PCplastocyanin PEPphosphoenolpyruvate PEPCphosphoenolpyruvate carboxylase PETCphotosynthetic elect ron transport chain PIBpost-illumination burst pmfProton motive force PNphotosynthetic rate PRNet photosynthetic rate PRKasephosphoribulokinase PrxRthylakoid-associ ated peroxiredoxins PGLP-2-phosphoglycerate phosphatase PKABP2-peroxo-3-keto-D-a rabitinol 1,5-bisphosphate PPDKorthophosphate dikinase, a C4 enzyme RCARubisco activase RD29A:LUCA marker gene responsive to dessication RLPRubisco-like protein ROSreactive oxygen species RuBPribulose 1,5-bisphosphate SAsalicyclic acid Sc/operfect specificity for CO2 over O2 SGATserine-glyoxylate aminotransferase SHMTserine hydroxymethyl transferase SODsuperoxide dismutase SR1The control line in a GS experiment TA 41 kDa monomeric protein re quiring THF cofactor from GDC THF5,6,7,8-tetrahydrofolate TIMA conserved protein fold with 8 -helices and 8 -parallel strands. TKTransketolase TPI-triose-phosphate isomerase TpFS-3/6Transgenic tobacco plants to e xpress a cyanobacterial FBPase/SBPase gene Trxthioredoxin tsrA gene that codes for tart ronic semidaldehyde reductase TSRtartronic semialdehyde Vcmaximum carboxylation rate Vo-maximum oxygenation rate Vc Ko / Vo KcSpecificity factor of Rubiso WTwild type

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