Chapter 12 With a Little Help from My Friends: The Central Role of Photorespiration and Related Metabolic Processes in the Acclimation and Adaptation of Plants to Oxygen and to Low-CO₂ Stress

Abstract

The photorespiratory repair pathway (photorespiration) is a supplement to the Calvin-Benson cycle that allows photosynthetic fixation of CO₂ to be based on the photochemical splitting of water , that is, in the presence of oxygen. Photorespiration is necessary because oxygen can replace CO₂ at the CO₂ fixation enzyme ribulose-1,5-bisphosphate carboxylase (Rubisco) and oxidize the CO₂ acceptor molecule ribulose-1,5-bisphosphate (RuBP), generating the powerful enzyme inhibitor 2-phosphoglycolate (2PG). 2PG must be removed because its accumulation would block operation of the Calvin-Benson cycle and in turn halt photosynthesis. Photorespiratory removal and detoxification of 2PG occurs by recycling to 3-phosphoglycerate which can be used to generate fresh RuBP. This repair process efficiently recovers three out of four misdirected carbon atoms while one carbon is oxidized and lost as CO₂.

Abbreviations: 2OG – 2-oxoglutarate; 2OGDH – 2OG dehydrogenase; 2PG – 2-phosphoglycolate; 3HP – Hydroxypyruvate; 3PGA – 3-phosphoglycerate; AOX – Alternative oxidase; BASS6 – bBile acid sodium symporter 6; CAT – Catalase; CCM – Carbon dioxide concentrating mechanism; Complex II – Succinate dehydrogenase; DiT1 – Dicarboxylate translocator (chloroplastic); Fd-GOGAT – Glutamate synthase (ferredoxin-dependent); FMN – Flavin mononucleotide; GCS – Glycine cleavage system; GGAT – Glutamate-glyoxylate aminotransferase; GLYK – Glycerate 3-kinase; GOE – Great Oxidation Event; GOX – Glycolate oxidase; GS2 – Glutamine synthetase (chloroplastic); HPR1 – NADH-dependent hydroxypyruvate reductase (peroxisomal); HPR2 – NADPH-dependent hydroxypyruvate reductase (cytosolic); MDH – Malate dehydrogenase; OA – Oxaloacetate; PAL – Present atmospheric level; PGLP – 2PG phosphatase; PLGG1 – Plastidal glycolate glycerate translocator 1; PLP – Pyridoxal 5-phosphate; Rubisco – RuBP carboxylase/oxygenase; RuBP – Ribulose 1,5-bisphosphate; SBPase – Sedoheptulose 1,7-bisphosphatase; SGAT – Serine-glyoxylate aminotransferase; SHMT – Serine hydroxymethyltransferase; THF – Tetrahydrofolate; TPI – Triosephosphate isomerase; UCP – Uncoupling protein (aspartate-glutamate carrier)

Summary

The photorespiratory repair pathway (photorespiration) is a supplement to the Calvin-Benson cycle that allows photosynthetic fixation of CO₂ to be based on the photochemical splitting of water , that is, in the presence of oxygen. Photorespiration is necessary because oxygen can replace CO₂ at the CO₂ fixation enzyme ribulose-1,5-bisphosphate carboxylase (Rubisco) and oxidize the CO₂ acceptor molecule ribulose-1,5-bisphosphate (RuBP), generating the powerful enzyme inhibitor 2-phosphoglycolate (2PG). 2PG must be removed because its accumulation would block operation of the Calvin-Benson cycle and in turn halt photosynthesis. Photorespiratory removal and detoxification of 2PG occurs by recycling to 3-phosphoglycerate which can be used to generate fresh RuBP. This repair process efficiently recovers three out of four misdirected carbon atoms while one carbon is oxidized and lost as CO₂. Photorespiration is a high-flux bearing pathway in most land plants that involves more enzymes than the Calvin-Benson cycle itself and in plants requires cooperation of different cellular compartments. It thereby represents one of the most complex examples of metabolic organisation and in many ways interacts with other metabolism, such as respiration, oxidative phosphorylation, nitrogen and sulphur assimilation. Photorespiration is also a very ancient pathway that likely evolved in the dual-photosystem bearing, oxygen-evolving photoautotrophic cyanobacteria at least 2.5–2.6 billion years ago. In this chapter, we will focus on recent advances concerning the metabolic integration and the regulation of photorespiration, its response to past and predicted future climate changes, and its potential for engineering more productive crops.

I. Introduction

The photorespiratory pathway (in short photorespiration) is best understood as a set of enzyme reactions that collectively prevent accumulation of the metabolic inhibitor 2-phosphoglycolate (2PG) produced in an idle side-reaction of the enzyme at the interface between inorganic and organic carbon, ribulose 1,5-bisphosphate (RuBP) carboxylase. Thereby, photorespiration is a repair pathway that is integral and essential to photosynthetic CO₂ fixation in all plants, algae and cyanobacteria. Historically, this section of metabolism was known as the ‘glycolate pathway’, while the term ‘photorespiration‘was initially used to describe the associated loss of CO₂ from photosynthesising leaves.

RuBP carboxylase is commonly known by the acronym Rubisco for RuBP carboxylase/oxygenase, indicating the promiscuous chemistry of the enzyme. The enzyme reaction starts with the reversible enolization of RuBP to produce a reactive 2,3-enediolate intermediate (Fig. 12.1). In fact, Rubisco evolved from a primordial enolase (Ashida et al. 2005). While the enediolate normally binds CO₂ to form two molecules of 3-phosphoglycerate (3PGA), it can also react with oxygen to produce one molecule each of 2PG and 3PGA (Bowes et al. 1971). The oxidation (‘metabolite damage’) of RuBP to 2PG and 3PGA is the most important of several side reactions catalysed by Rubisco. Its rate is determined by a number of factors including Rubisco amount and kinetic properties and the concentrations of RuBP, oxygen and CO₂ (Laing et al. 1974; Peisker 1974). Even though Rubisco strongly favours CO₂ over oxygen, the 2PG biosynthetic rate is enormous particularly in most land plants. This is because the concentration of oxygen very much exceeds that of CO₂ in the atmosphere (21% oxygen versus 0.04% CO₂ in present air) and particularly in photosynthesizing cells. Thus, RuBP oxidation has three very direct implications for photosynthesis and hence plant growth: competitive inhibition by oxygen of CO₂ fixation, non-productive consumption of RuBP, and the inhibition of RuBP regeneration in the Calvin-Benson by 2PG. This corresponds to the fact that plants cannot thrive in air and require artificial conditions to survive when 2PG degradation is blocked (Somerville and Ogren 1979; Schwarte and Bauwe 2007). Removal of 2PG in the photorespiratory pathway involves that two molecules of 2PG are converted to one molecule of 3PGA, scavenging 3 out of four carbon atoms that were misdirected to 2PG, while only one carbon atom becomes reoxidized to CO₂ (Tolbert 1997). Only about half of the re-liberated CO₂ can be reassimilated particularly in C₃ plants, whereas a very considerable fraction is lost to the atmosphere. This feature explains why photorespiration was long misunderstood as wasteful process that should be eliminated in order to improve photosynthesis and crop yields.

Fig. 12.1.

CO₂ fixation by Rubisco. The enediolate form of RuBP can be carboxylated leading to the formation of two molecules 3PGA, or it can be oxidized to form one molecule each of 2PG. Under present atmospheric conditions, about every third RuBP would become oxidized, producing two molecules of 2PG carrying four previously fixed carbon atoms (upper part). At the same time, carboxylation of RuBP can only fix about four CO₂ molecules as 3PGA (lower part). In sum, all freshly fixed carbon becomes locked in 2PG and can be released only by operation of the photorespiratory pathway. (Modified after Bauwe et al. (2012)

Overall, the photosynthetic-photorespiratory supercycle formed from the Calvin-Benson cycle and its photorespiratory supplement belongs to the key inventions of evolution, being responsible for nearly all biological CO₂ fixation on Earth. It is also a very ancient metabolic process that evolved together with the ability to use water , instead of hydrogen sulphide, as the electron donor for CO₂ fixation in photosynthetic cyanobacteria about 2.5–2.6 and possibly much earlier. Looking into the future, given the sheer magnitude of photorespiration in C₃ plants and its multiple interactions particularly with the Calvin-Benson cycle but also with mitochondrial energy metabolism and many other processes, it is not surprising that photorespiration has become a key target for breeders in order to generate crops with better photosynthetic performance and higher yield.

In this chapter, we will focus on recent advances concerning the metabolic integration and the regulation of photorespiration, the response of photosynthetic-photorespiratory metabolism to past and predicted future climate changes, and its potential for engineering more productive crops. This article is in part based on two earlier reviews (Hagemann and Bauwe 2016; Bauwe 2018). We additionally recommend reviews on Rubisco (Bracher et al. 2017), metabolite transport (Eisenhut et al. 2015), metabolic interactions (Abadie et al. 2017; Heyneke and Fernie 2018), and methods and protocols relevant for photorespiration research (Fernie et al. 2017) for more information on individual topics.

II. A Bird’s-Eye View at the Core Pathway

Plant photorespiration requires ten different enzyme reactions in the core pathway and a number of auxiliary enzymes for additional processes, such as ammonia refixation. In land plants, these enzymes are distributed over four subcellular compartments: the chloroplast, the peroxisome (lost in green algae), the mitochondrion and the cytosol (Fig. 12.2). Rubisco triggers but, by definition, is not part of the repair pathway. When assessed by the number of participating enzymes and subcellular compartments, photorespiration is more complex than the Calvin-Benson cycle and indeed represents one of the most complex examples of metabolic organisation in eukaryotes. Central to photorespiration is the conversion of the two-carbon skeleton of 2PG into a three-carbon skeleton, 3PGA, which is compatible with the Calvin-Benson cycle.

Fig. 12.2.

The plant photorespiration core pathway (light blue) spans three organelles: the chloroplast, the peroxisome, and the mitochondrion. The enzymes are PGLP, GOX, GGAT, SGAT, GCS (comprising three enzymes), SHMT, HPR1, and GLYK. Known transporters are PLGG1 and BASS6. CAT detoxifies the generated hydrogen peroxide. The GS-GOGAT cycle (green) re-fixates the ammonia released by GCS. Complex I and several shuttle systems (light grey underlay) keep the redox balance. At least two enzymes of the core pathway can be circumvented in specific conditions. First, the cytosolic HPR2 bypass supports HPR1 when the peroxisomal MDH does not provide NADH rapidly enough for hydroxypyruvate reduction. Second, ATP consumption by cGLYK in the cytosol of shade-grown plants helps alleviating photoinhibition of chloroplasts. Note this scheme is adapted from Fernie and Bauwe, Plant Journal submitted

A. 2-Phosphoglycolate Is Dephosphorylated in the Chloroplast

First, still within the chloroplast, 2PG phosphatase (PGLP) removes the phosphate, producing glycolate. This tetrameric enzyme belongs to the large family of haloacid dehalogenase-type phosphatases and requires Mg²⁺ for activity. An additional, cytosolic PGLP is not involved in photorespiration (Schwarte and Bauwe 2007) but serves to destroy toxic side products from mainline carbon metabolism (Collard et al. 2016).

Glycolate moves out of the chloroplast through two recently identified transporters, the glycolate-glycerate antiporter PLGG1 (Pick et al. 2013) and the glycolate transporter BASS6 (South et al. 2017). Partnering of the two transporters, with and without co-transport of glycerate in the opposite direction, balances photorespiratory carbon flow in a way that two glycolate molecules are exported per one imported glycerate. Plants deficient in both transporters are still viable in air (South et al. 2017), which confirms earlier reports that glycolic acid and glyceric acid to some extent can diffuse through the chloroplast envelope.

B. Glycolate Is Converted to Glycine in the Peroxisome

Glycolate enters the peroxisome through a protein pore, likely PMP22, (Reumann 2011), and becomes oxidised to glyoxylate followed by transamination to glycine. This amino acid is central to the photorespiratory pathway because, later in the mitochondria, it can serve as a one-carbon donor and as a one-carbon acceptor to produce one molecule of serine from two molecules of glycine.

First, the flavin mononucleotide (FMN)-dependent enzyme glycolate oxidase (GOX; also known as short-chain L-2-hydroxy acid oxidase)) oxidizes glycolate to glyoxylate, using molecular oxygen as the terminal oxidant and generating hydrogen peroxide. The large amount of hydrogen peroxide produced within the peroxisome must be rapidly removed because it can cause oxidative DNA damage and trigger peroxisome-associated protein degradation. Decomposition occurs by a Fe heme-containing homotetrameric monofunctional catalase (CAT), an ancillary enzyme that has one of the highest turnover numbers of all enzymes and forms part of a general scavenging network for reactive oxygen species.

Transamination of glyoxylate to form glycine requires two enzymes, glutamate-glyoxylate aminotransferase (GGAT) and serine-glyoxylate aminotransferase (SGAT). Given their low affinity to glycine these enzymes catalyse physiologically irreversible reactions and potentially can operate with a range of different substrates. GGAT uses glutamate as the amino donor, which at the same time generates 2-oxoglutarate (2OG) needed for the refixation of photorespiratory NH₃ in the GS-GOGAT cycle discussed further below. The second aminotransferase, SGAT, simultaneously acts on the two-carbon and the three-carbon branch of the pathway: It transfers the serine amino group to glyoxylate, producing glycine and hydroxypyruvate (3HP). SGAT can also work with asparagine (Zhang et al. 2013), which potentially links photorespiration with general N metabolism (Modde et al. 2017). It is hypothesized that glyoxylate escaping transamination under stress conditions is scavenged by a pair of high-affinity glyoxylate reductases, the cytosolic GLYR1 and the plastidial GLYR2 (Allan et al. 2009; Zarei et al. 2017).

C. Mitochondrial Enzymes Convert Glycine to a Three-Carbon Compound, Serine

The path on which photorespiratory metabolites enter and exit the mitochondrion is currently unknown, but inter-organellar connections are clearly facilitated by organellar extensions as they also exist between chloroplasts and peroxisomes (termed matrixules, stromules, and peroxules, Mathur et al. 2012). Exchange through the outer membrane is thought to rely on passive diffusion through voltage-gated channels (also called mitochondrial porins) in the outer membrane (for example Schell and Rutter 2013). Movement of glycine through the inner mitochondrial membrane seems to be more restrictive and likely involves diffusion and carrier mediated transport (reviewed in Eisenhut et al. 2013a).

Within the mitochondria, the central function of the photorespiratory pathway takes place: conversion of the two-carbon compound glycine (produced from 2PG) into the three-carbon compound serine (to finally form 3PGA). This requires the multienzyme glycine cleavage system (GCS, in plants also known as the glycine decarboxylase complex) and serine hydroxymethyltransferase (SHMT; reviews in Kikuchi et al. 2008; Schirch and Szebenyi 2005). These enzymes are ubiquitous components of one-carbon metabolism in all plant cells; however, they are highly abundant in the matrix of green leaf mitochondria, resulting in the association of the four GCS proteins to form an as yet poorly characterized fragile multiprotein complex (Neuburger et al. 1986; Oliver et al. 1990).

The GCS reaction cycle comprises three reactions, which are catalysed by the pyridoxal 5-phosphate (PLP)-dependent enzyme P-protein (glycine decarboxylase), the polyglutamyl tetrahydrofolate (THF)-dependent enzyme T-protein (aminomethyltransferase), and the NAD⁺-dependent enzyme L-protein (dihydrolipoamide dehydrogenase). The lipoic acid-containing H-protein (hydrogen carrier protein), the fourth GCS protein, interacts as a shared substrate successively with the P-, T-, or L-protein to transfer reaction intermediates and reducing equivalents bound to its lipoyllysine arm from one enzyme to the other and finally to NAD⁺. The entire process requires one molecule each of glycine, tetrahydrofolate (THF), and NAD⁺ to produce one molecule of methylenetetrahydrofolate (CH₂THF). In addition, one molecule each of CO₂ and ammonia are released (by P- and T-protein, respectively) and NADH is produced (by L-protein). The mitochondrial glutamate transporter BOUT DE SOUFFLE (BOU) could be important for THF polyglutamylation (Porcelli et al. 2018). To provide the GCS with sufficient fresh NAD⁺, much of the generated NADH is rapidly reoxidized in a process that is, to some extent though not completely, coupled to adenosine triphosphate (ATP) synthesis (Gardeström and Igamberdiev 2016). That way photorespiration is a major supplier of NADH to daytime oxidative phosphorylation. The remaining NADH is exported to the cytosol via malate shuttles (Selinski and Scheibe 2019). For these reasons, photorespiratory metabolism has a strong effect on the cellular NADH/NAD⁺ balance and influences other cellular processes such as sucrose synthesis, the tricarboxylic acid (TCA or Krebs) cycle and nitrate assimilation.

Next, the PLP-dependent enzyme SHMT combines CH₂THF with a second molecule of glycine to make serine and regenerate THF for the GCS. It was found that SHMT also produces considerable amounts of 5-formyl THF. This compound strongly inhibits SHMT and must therefore continually be detoxified (Collakova et al. 2008), adding a second-level metabolic repair system on top of photorespiration (Fig. 12.3).

Fig. 12.3.

Photorespiration requires several secondary-level repair pathways. For example, SHMT and other enzymes are inhibited by 5-formyl THF, which is produced in considerable amounts by SHMT itself (shown in red). Detoxification and recycling to THF (black route) requires four folate-interconverting enzymes, 5,10-CH₂THF dehydrogenase combined with 5,10-methenyl THF cyclohydrolase in a bifunctional enzyme (DHC), 5-formyl THF cycloligase (5-FCL) and 10-formyl THF deformylase (10-FDF). Formate dehydrogenase (FDH) oxidizes the produced formate to CO₂. If the photorespiratory THF cycle is blocked, plants accumulate massive amounts of glycine and cannot survive in normal air (Collakova et al. 2008). Note this scheme is adapted from Bauwe (2019)

D. Back in the Peroxisome, Hydroxypyruvate Is Produced from Serine and Becomes Oxidized to Glycerate

Serine goes back into the peroxisomes, where it delivers its amino group to glyoxylate to produce glycine and hydroxypyruvate, the latter of which becomes reduced to glycerate by NADH-dependent hydroxypyruvate reductase (HPR1; Givan and Kleczkowski 1992) . This enzyme, together with CAT, GOX, and NAD⁺-malate dehydrogenase (MDH), is one of the four major proteins in leaf peroxisomes. NADH cannot permeate the peroxisomal membrane and must be provided by peroxisomal malate dehydrogenase (pMDH), which oxidizes malate imported from the cytosol.

In contrast to the deleterious effect of blocks in other reactions of the pathway, neither the deletion of HPR1 (Murray et al. 1989; Timm et al. 2008) nor that of pMDH (Cousins et al. 2008) strongly impairs plant growth. This is because hydroxypyruvate can exit the peroxisome and alternatively be reduced to glycerate in the cytosol by the auxiliary enzyme NADPH-dependent hydroxypyruvate reductase 2 (HPR2; Timm et al. 2011; Ye et al. 2014). Hydroxypyruvate flux through the cytosol is small in moderate environments and likely defined by the rate of NADH supply to HPR1. It is speculated that the bypass allows more flexibility in the short-term adaptation of photorespiratory metabolism to changing environmental conditions (Givan and Kleczkowski 1992; Timm et al. 2011).

E. Phosphorylation of Glycerate to 3PGA Completes the Photorespiratory Pathway

Glycerate enters the chloroplast in exchange for glycolate through PLGG1 and to some extent possibly by diffusion of glyceric acid. In the chloroplasts, and in some conditions also the cytosol (Ushijima et al. 2017), D-glycerate 3-kinase (GLYK; Kleczkowski and Randall 1983; Boldt et al. 2005) completes the photorespiration pathway by returning three out of four 2PG carbon atoms that were initially misdirected to 2PG back to the Calvin-Benson cycle in the form of 3PGA. GLYK is the only glycerate kinase that produces 3PGA. Most bacteria and animals do not use GLYK but a glycerate 2-kinase, which produces 2PGA (Bartsch et al. 2008; Kern et al. 2011).

III. Photorespiration Interacts with Other Metabolism and Requires Secondary-Level Repair Pathways

A. Regulatory Interaction with the Calvin-Benson Cycle

The photorespiratory pathway obviously is most intimately connected to the Calvin-Benson cycle; however, the interaction goes beyond the mere recycling of 2PG to 3PGA. For example, studies with mutants and chemical inhibitors have consistently shown that any impairment of the photorespiratory pathway compromises photosynthesis (discussed in Timm et al. 2016), but we do not yet fully understand the molecular mechanisms of how photosynthesis is inhibited in this circumstances. Related to this and even more important is the fact that we do not know much about the significance of such interaction for the regulation of photosynthesis in natural conditions.

In cyanobacteria, the prokaryotic inventors of photorespiration, 2PG acts as a low-CO₂ signal that serves to coordinate carbon and nitrogen metabolism (Jiang et al. 2018). At low CO₂, more 2PG binds to two transcriptional regulator proteins: the transcriptional activator CmpR and the transcriptional repressor NdhR. The resulting conformational changes promote dissociation of NdhR from and association of CmpR with their target DNA, increasing the expression of photosynthesis-related genes. NdhR can also bind 2OG, which acts as a corepressor. Therefore, 2PG (sensing CO₂ starvation) and 2OG (sensing nitrogen starvation) function together as fine sensors for the coordination of carbon and nitrogen metabolism in cyanobacteria (Jiang et al. 2018; Zhang et al. 2018). Because of the restriction of 2PG to the chloroplast, it is not likely this kind of transcriptional control occurs in eukaryotic phototrophs.

2PG can however also signal changes of the CO₂ concentration at the biochemical level. This is possible because 2PG very efficiently inhibits the activity of SBPase and TPI, already at μM levels (Anderson 1971; Flügel et al. 2017). Varying inhibition of these enzymes in response to short-term changes in the environment in turn modulates the rate of RuBP regeneration and hence the activity of the Calvin-Benson cycle as a whole, including RuBP oxygenation (that is, photorespiration) and starch synthesis. This type of biochemical regulation by 2PG indeed occurs in plants as demonstrated by the better photosynthetic performance and faster growth observed after PGLP overexpression (Flügel et al. 2017). Notably, inhibition of the chloroplastidal TPI can to some extent be bypassed by the export of glyceraldehyde 3-phosphate to the cytosol where it is converted to glucose 6-phosphate (G6P). The produced G6P is re-imported into the chloroplast and refills the Calvin-Benson cycle, either by direct conversion to fructose 6-phosphate or by oxidative decarboxylation to ribulose 5-phosphate, the glucose 6-phosphate shunt (Sharkey and Weise 2016; Li et al. 2019).

While there is little direct evidence yet, it appears that photorespiratory metabolites which in contrast to 2PG equilibrate between different subcellular compartments, such as glycine and serine (Timm et al. 2013; Modde et al. 2017) or glyoxylate and glycolate (Leegood et al. 1995), are also involved in the regulation of photosynthesis. For example, overexpression in Arabidopsis (Arabidopsis thaliana) of the GCS H-protein alone (Timm et al. 2012a) or in combination with the chloroplastidal enzyme SBPase (Simkin et al. 2017; tobacco, Lopez-Calcagno et al. 2018) produced plants with increased rates of net-CO₂ assimilation and plant growth. Similar growth effects were observed with plants overexpressing the GCS L-protein (Timm et al. 2015). All this shows that the facilitation of carbon flux through the mitochondrial section of the photorespiratory pathway stimulates photosynthetic CO₂ assimilation in the Calvin-Benson cycle. The molecular players in this inter-organellar crosstalk remain to be identified. In addition to the metabolite-level control of enzyme activities, it appears that transcriptional, post-transcriptional and post-translational processes are likewise important.

B. Photorespiration and Photoinhibition

The interplay between photorespiration, photosynthetic electron transport and photoinhibition of PS II has long been controversially debated. In hindsight, the magnitude of the contribution of photorespiration to photoprotection may have been overemphasized as the result of the aspiration to ascribe a reasonable function to the supposedly ‘wasteful’ process of photorespiration. At present, there is a consensus that plants have a number of much more efficient mechanisms by which to protect their photosynthetic apparatus from photoinhibition under natural conditions (reviewed in Derks et al. 2015), but photorespiration contributes in specific circumstances.

First, photorespiration consumes ATP and reducing power, providing an alternative electron sink when CO₂ is not sufficiently available. This feature can gain some relevance when plants suffer from a severe water deficit and the stomata of the leaves close, which at the same time restricts the entry of CO₂ (for example Osmond et al. 1997).

Second, the rapid photoinhibition that can be observed in many mutants of the photorespiratory pathway at low CO₂ has been attributed to translational suppression of the de novo synthesis of the D1 protein, which in turn inhibits the repair of photodamaged PS II (Takahashi et al. 2007). Repair of PS II requires large amounts of ATP (Murata and Nishiyama 2018), which links this finding to the unexpected demonstration that photorespiration at least in some conditions does not decrease but increase the chloroplastidal ATP level (Ushijima et al. 2017). These authors found that, due to phytochrome-mediated alternative promotor selection, shade-grown Arabidopsis plants accumulate a cytoplasmic isoform of GLYK, cytGLYK. This isoform alone – without the plastidic GLYK – is able to some extent drive photorespiration and represents another cytosolic photorespiratory bypass. Photorespiratory ATP consumption on the cytosolic route lowers the ATP consumption by photorespiration in the chloroplast and alleviates photodamage induced by fluctuating high light, which, by contrast to constant light used in many studies (Apelt et al. 2017), is the standard day condition for plants or individual leaves in many ecosystems (Slattery et al. 2018).

Finally, photorespiration can also interfere with photosynthetic electron transport. One example is the better light-use efficiency and the significantly higher leaf ATP levels of GCS L-protein overexpressors (Timm et al. 2015), which points to the possibility of crosstalk between photorespiration and cyclic electron flow around photosystem I. Whilst this hypothesis still needs to be tested, it was shown that glycolate can replace bicarbonate from the non-heme iron at the acceptor side of photosystem II, lowering the production of singlet oxygen species and downregulating photosynthetic electron transport and maybe the synthesis of RuBP synthesis in the Calvin-Benson cycle (Messant et al. 2018).

C. Photorespiration and Stomatal Regulation

A further interaction between photorespiration and photosynthesis of note is the early observation that α-hydroxysulfonates which act as competitive inhibitors of GOX prevent the opening of stomata in the light in a manner that is consistent with the levels of glycolate in tobacco (Zelitch and Walker 1964). Furthermore, the authors demonstrated in the same manuscript that CO₂-induced closing of stomata can be reversed on the provision of glycolate to the leaf discs. In support of this a recent study identified that a mutant allelic to plgg1-1 demonstrated the involvement of this transporter in abscisic acid (ABA )-mediated stomatal movement, however, mechanistic details underlying this phenomena are yet to be resolved (Dong et al. 2018). Moreover, it is currently unclear to what extent such a regulation will interact with the influence on stomatal movement known to be exerted by sugars and organic and fatty acids (Daloso et al. 2017).

D. Metabolite Shuttles

Photorespiration is a major highway of plant metabolism and likely tightly regulated on multiple levels (Leegood et al. 1995; Bauwe et al. 2010; Keech et al. 2017). Particularly, photorespiration produces and consumes in equimolar amounts large amounts of NADH, but this occurs in different organelles and is not directly coupled. Moreover, an estimated two-thirds of the NADH generated during glycine decarboxylation is immediately reoxidized in the mitochondrial oxidative respiratory chain, which ensures rapid regeneration of NAD⁺ to drive rapid glycine decarboxylation and produces ATP for sucrose synthesis and nitrate reduction in the cytosol (reviewed in Gardeström and Igamberdiev 2016).

The remaining third of reducing equivalents, if not dissipated in the alternative respiratory pathway, is exported to the cytosol via two shuttle systems (Fig. 12.2). One shuttle, the mitochondrial ‘malate valve’, exchanges malate for oxaloacetate (OA), which requires mitochondrial and cytosolic isoforms of malate dehydrogenase (MDH) and one or several dicarboxylate translocators (DIC; Hanning et al. 1999; Palmieri et al. 2008). The second system is a malate-aspartate shuttle, which keeps OA levels low and involves an exchange of glutamate for 2OG to ensure the nitrogen equilibrium between the two compartments. It requires compartment-specific isoforms of MDH and aspartate-glutamate aminotransferase (ASAT) and one or two mitochondrial transporter(s) of aspartate, glutamate, and dicarboxylates that were recently identified (Monné et al. 2018) and previously known as mitochondrial uncoupling proteins (UCP) 1 and 2 (Sweetlove et al. 2006).

In any case, the exported fraction of reducing equivalents does not match the needs of photorespiratory HP reduction in the peroxisome and is complemented from the chloroplast (Voon et al. 2018). It was long thought that this occurs by malate export from the chloroplast (the chloroplast ‘malate valve’; reviewed in Selinski and Scheibe 2019) with the vacuole serving to temporarily store excess malate (Szecowka et al. 2013), but the actual situation is less clear. For example, full inactivation of neither the peroxisomal pMDH (Cousins et al. 2008), nor the chloroplastidal malate valve enzyme NADP-MDH (Hebbelmann et al. 2012) had any substantial effect on photorespiration. Recent work has meanwhile proven that the plastidial NAD-dependent MDH has a moonlighting role in early chloroplast development through its interaction with an FtsH12-FtsHi protease complex that is more important than any role in redox regulation (Schreier et al. 2018). Moreover, the combined inactivation of HPR1 and pMDH also did not produce the characteristic phenotype of a photorespiratory mutant (Pracharoenwattana et al. 2010; Cousins et al. 2011). Therefore, it appears that not a single process but rather a multi-pathway network including proteins of as yet unknown function (Wang et al. 2018) provides NADH to the cytosol at daytime. Recent surveys appear to have provided comprehensive information concerning the plastidial complement of metabolite transporters (Facchinelli and Weber 2011) and functional and evolutionary analysis of these proteins will likely provide highly insightful in the identification of other proteins mediating inter-organellar redox transfer. Actually, it appears that indeed several bypasses complement the core photorespiratory pathway, including a 2PG-triggered glucose 6-phosphate shunt that releases additional “photorespiratory” CO₂, explaining the extraordinary effect of oxygen on the CO₂ compensation point of plants without peroxisomal hydroxypyruvate reductase (Li et al. 2019).

Recent evidence for the role of two further transporters in, or associated with, photorespiratory metabolism has been provided (Eisenhut et al. 2013b). The loss-of-function of the first of these – an endoplasmic reticulum ATP antiporter – was demonstrated to exhibit a photorespiratory phenotype although the exact mechanistic reason underlying this remains to be uncovered (Hoffmann et al. 2013). Loss of function of the mitochondrial glutamate transporter A BOUT DE SOUFFLE (BOU), which is necessary for the generation of polyglutamylated THF, is also associated with defective photorespiratory metabolism and is required for meristem growth at ambient CO₂ (Eisenhut et al. 2013b; Porcelli et al. 2018) as well as being important for sulphur assimilation and its cross talk with carbon and nitrogen metabolism in Arabidopsis (Samuilov et al. 2018).

E. Nitrogen Metabolism

Photorespiration provides glycine and serine for a number of biosynthetic pathways in photosynthesising cells, including the synthesis of cysteine and glutathione, proteins and the many compounds which require one-carbon units for their biosynthesis. This efflux is small relative to total photorespiratory flux though (Abadie et al. 2016). By contrast, reassimilation of the NH₃ released in GCS T-protein reaction happens in a high-flux bearing pathway that operates with very high efficiency: not more than about 0.01% of photorespiratory NH₃ is lost from the leaf (Mattsson et al. 1997). The process requires ammonia transport across organellar membranes to the chloroplast, possibly through aquaammoniaporins (Jahn et al. 2004; Li et al. 2017). Import of 2OG into the chloroplasts and the export of glutamate is accomplished by two translocators (Woo et al. 1987). The bifunctional dicarboxylate translocator (DiT1), which is identical with the chloroplastic malate valve mentioned above, exchanges 2OG or oxaloacetate for malate (Kinoshita et al. 2011). DiT2.1 exchanges Glu for malate in the opposite direction (Renné et al. 2003), which at the same time balances the opposed malate flow through DiT1. Once in the chloroplast, the ATP-dependent glutamine synthetase (GS2) binds the ammonia to glutamate forming glutamine. Next, the ferredoxin-dependent glutamate synthase (Fd-GOGAT) transfers the amide-nitrogen from glutamine to 2-oxoglutarate to yield two molecules of glutamate, which can be used as a general amino donor including glycine synthesis by GGAT or enters a new round of ammonia (re-)fixation. This GS-GOGAT cycle is a secondary-level repair pathway concerning the reassimilation of photorespiratory ammonia, but it is also essential for nitrate assimilation and the central link between carbon and nitrogen metabolism (Zhang et al. 2018).

The high interdependence of the processes of photorespiration, nitrate assimilation, and mitochondrial respiration was yet further illustrated in the evaluation of transgenic tomato (Solanum lycopersicum) plants deficient in the expression of the mitochondrial isoforms of citrate synthase and isocitrate dehydrogenase, where both sets of plants displayed compromised nitrate assimilation and altered rates of photorespiration (Sienkiewicz-Porzucek et al. 2008; Sienkiewicz-Porzucek et al. 2010). Corresponding to these experimental findings, elementary flux mode analysis suggests that photorespiratory metabolism exists in multiple forms, a subset of which is stoichiometrically coupled to nitrate reduction. (Huma et al. 2018).

Some controversy remains though. Firstly, a report suggesting dual targeting of GS2 to both chloroplasts and mitochondria (Taira et al. 2004), with the accumulation in mitochondria having a regulatory rather than enzymatic function, provoked the postulation of several functional modes of photorespiratory ammonia refixation (Linka and Weber 2005). More convincing was the demonstration that photorespiratory SHMT activity requires the mitochondrial accumulation of Fd-GOGAT (Voll et al. 2006), but, secondly, it is not known why this is necessary and how it occurs. Conversely, antisense reduction of SHMT activity resulted in a shift in the diurnal ammonium metabolism in potato (Solanum tuberosum) leaves indicating that internal accumulation of post-photorespiratory ammonia leads to nocturnal activation of GS2 and Fd-GOGAT (Schjoerring et al. 2006). Thirdly, it is controversially debated whether, or how much and by which mechanisms, lower photorespiration as induced by elevated atmospheric CO₂ interferes with nitrate assimilation (Bloom and Lancaster 2018; Busch et al. 2018; Huma et al. 2018; Andrews et al. 2019; Bloom et al. 2019).

F. TCA Cycle, Respiratory Electron Transport Chain, Oxidative Phosphorylation

The links between photorespiration and day respiration are supported by considerable cumulative evidence and highly important for several reasons (Obata et al. 2016; Tcherkez et al. 2017). Firstly, photorespiration is the highest flux bearing process in mitochondria under photosynthetic conditions. Secondly, the photorespiratory influx of reducing equivalents via glycine very much exceeds any realistic rate of daytime ATP production by oxidative phosphorylation, but the NADH produced during glycine decarboxylation nevertheless must be rapidly re-oxidized to NAD⁺ in order to provide further oxidant for glycine decarboxylation and to maintain the mitochondrial redox balance. Thirdly, glycine oxidation and the TCA cycle potentially compete for available NAD⁺. These conflicts are balanced by various mechanisms operating on the levels of (i) downregulation of the TCA cycle as a major competitor for NAD⁺, (ii) the mitochondrial electron transport chain and oxidative phosphorylation, (iii) outsourcing NADH oxidation to other cellular compartments by shuttle mechanisms as discussed above.

Downregulation of the TCA cycle occurs by the phosphorylation of the mitochondrial PDH (Budde and Randall 1990), redox regulation of other TCA cycle enzymes (Daloso et al. 2015) and metabolite-level regulation particularly by ammonia and NADH (Nunes-Nesi et al. 2013). Collectively, these changes not only downregulate but also re-organize carbon flow through the TCA cycle at daytime in a way that it is not a cyclic process anymore because the conversion of 2OG to succinate becomes drastically reduced or even stops in the light, while malate and fumarate pools build up (Tcherkez et al. 2009; Tcherkez et al. 2012; Lee and Millar 2016). The resulting ‘horseshoe” structure of the TCA ‘cycle’ at daytime comprises a reductive branch (producing malate and fumarate, consuming NADH) and an oxidative branch (producing 2OG, generating NADH). Together, this helps to balance the consumption and production of NADH in the photorespiring mitochondrion, alleviating competition with glycine oxidation. The daytime mode of the TCA cycle also reminds to the evolutionary origin of the pathway, where the addition of just one enzyme, succinyl-CoA synthetase, may have combined two different linear pathways (one for amino acid and one for heme synthesis) into a cyclic, potentially reversible, multi-functional process (Meléndez-Hevia et al. 1996).

It is not yet known how the 2OG dehydrogenase (2OGDH) and/or succinyl-CoA synthetase and/or the succinate dehydrogenase (Complex II) are down-regulated in the light. Recently, thioredoxin o-mediated redox regulation of Complex II and possibly also of succinyl-CoA synthetase was demonstrated (Daloso et al. 2015). By these not yet fully understood changes, 2OG synthesis is retained to adequately serve nitrate assimilation and dynamic changes in the photorespiratory nitrogen flux but becomes separated from malate metabolism in the light.

Indeed, photorespiratory mutants are generally characterized as exhibiting altered levels of TCA cycle intermediates. Intriguingly, succinate and γ-aminobutyric acid (GABA) levels displayed very similar changes in mutants of the core photorespiratory pathway (Florian et al. 2013) including single and multiple mutants of HPR1, 2 and 3 (Timm et al. 2008; Timm et al. 2011; Timm et al. 2012b) and PGLP1 (Timm et al. 2012b) but not in mutants indirectly related to transport activities effecting photorespiration such as mitochondrial UCP1 (Sweetlove et al. 2006; Monné et al. 2018), plastidial MDH (Cousins et al. 2008) and a GOX overexpressing line (Fahnenstich et al. 2008). Similarly, although the function of the carbonic anhydrase subunit of NADH dehydrogenase (Complex I) is still under debate, a recent study of an Arabidopsis line deficient in the expression two carbonic anhydrase like gene also indicates the relationship between respiration and photorespiration (Fromm et al. 2016). Indeed, the metabolite profile of this mutant was characterized by lower accumulation of glycine and succinate and lower levels of citrate, malate and fumarate (Fromm et al. 2016). Also of note in this context is the assessment of the metabolite levels of the plastidial GS2 mutant (Pérez-Delgado et al. 2015). This mutant accumulates higher levels of ammonium, glutamine and 2OG concomitant to the accumulation of glycine and serine (Pérez-Delgado et al. 2015). When taken together, these findings provide remarkably conserved changes indicating that the interaction of photorespiration and respiration is largely co-ordinated by the reactions surrounding 2OG with those being involved either in ammonium assimilation (Xu et al. 2012; Zhang et al. 2018) or the GABA shunt (Fait et al. 2008). Intriguingly, photorespiratory metabolites were conversely altered in oxoglutarate dehydrogenase antisense lines (Araujo et al. 2012), confirming the close relationship between 2OG and photorespiration. Intriguingly, when photorespiratory rates were estimated from gas exchange measurements, they were found to increase in citrate synthase and aconitase lines but decline in succinyl-CoA synthetase and succinate dehydrogenase lines (Obata et al. 2016). This observation may be explained by the fact that the former two, but not the latter two enzymes, have extra-mitochondrial isoforms. However, whatever the explanation, this finding hints to the complexity of the relationship between the TCA cycle and photorespiratory pathway.

Furthermore and perhaps unsurprisingly given the intimate connection of the TCA cycle and oxidative phosphorylation mediated by the mitochondrial electron transport chain, other aspects of oxidative phosphorylation are also intertwined with photorespiration. Indeed the mutants of Complex I, ndufs4 and ndurfv1, are characterized by altered levels of both TCA cycle intermediates and photorespiratory intermediates (Kühn et al. 2015). Similarly, inhibition of mitochondrial ATP synthase by oligomycin treatment and the suppression of the δ-subunit of the ATP synthase complex also led to accumulation of serine and glycine (Geisler et al. 2012). Dissipation of the protein gradient across the mitochondrial inner membrane mediated by UCP1 has also been proposed to play a crucial role in maintaining the redox poise of the mitochondrial electron transport chain to facilitate photosynthetic metabolism. As mentioned above, the ucp1 mutant exhibits decreased levels of glycine and serine and reduced in vivo rates of glycine to serine conversion (Sweetlove et al. 2006). Alternative oxidases (AOXs) similarly mediate the dissipation of reducing equivalents by a route that is uncoupled from ATP production (Del-Saz et al. 2018); however, the aox1a mutant of Arabidopsis, which lacks the most abundant isoform of AOX, exhibits no change in the levels of photorespiratory, respiratory or GABA shunt metabolites under normal growth conditions (Giraud et al. 2008). Nevertheless, this mutant accumulates more glycine, pyruvate and citrate, and less serine than the wild-type plant when the electron transport via cytochrome oxidase is inhibited by antimycin A treatment (Strodtkötter et al. 2009). Under such conditions, the aox1a mutant also enhances the expression of the GCS P-protein (Strodtkötter et al. 2009). The interpretation of this study is however not very clear due to the potential effect of antimycin A particularly on the PGR5/PGRL1-dependent pathway of cyclic electron transport around photosystem I (Yamori and Shikanai 2016). Type II NAD(P)H dehydrogenases are similarly considered to function in the oxidation of NAD(P)H without producing ATP. Two studies characterized the metabolite profiles of RNAi-based suppression mutants of NDA1/NDA2 and NDB1, which are located on the matrix-faced and external surfaces of the mitochondrial inner membrane, respectively (Wallström et al. 2014a; Wallström et al. 2014b). Although the metabolic relationship with photorespiration is less clear for these type II dehydrogenases than it is for UCP and AOX, NDA suppression lines showed increased levels of glycine and serine under high-light conditions, while serine levels were reduced in the NDB line (Wallström et al. 2014a; Wallström et al. 2014b). Altogether, these results indicate a close interaction of mitochondrial electron transport and photorespiration, most likely due to the demand of recycling of NADH to NAD⁺ in order to support adequate photorespiratory flux (discussed in Bykova et al. 2014). A further point to note is the likely regulation of the GCS by thioredoxin (Buchanan and Balmer 2005), since this may represent a further common regulatory mechanism.

The examples above all describe molecular genetic analysis of the interactions of photorespiration and aspects of respiration; however, a wealth of studies have characterized changes in both processes following environmental perturbations. Whilst early studies focussed on the response of individual pathways, two recent studies specifically employed environmental perturbations anticipated to alter the photorespiratory-to-photosynthetic flux ratio in Arabidopsis and surveyed the cellular response at the metabolomic level. In the first of these, Arabidopsis plants growing under normal conditions were transferred to different light and temperature conditions anticipated to affect these parameters (Florian et al. 2014a). Interestingly, their results revealed similar behaviour in response to both treatments – especially with regard to photorespiratory intermediates – and suggest that these metabolic shifts are not mediated at the level of transcription. The second study took the same strategy but rather modified the CO₂ and oxygen partial pressures. Elevated CO₂ provoked the expected decrease in photorespiratory intermediates but also of the organic acids succinate, fumarate and malate and altered amino acid profiles (Florian et al. 2014b). By contrast, increasing oxygen had little effect on the rate of photorespiration but did lead to a rapid increase in photorespiratory metabolite levels but few other clear metabolic changes. However, when analysed alongside one another these two studies clearly reflect the metabolic interactions defined above. Four further papers are of interest in this vein, three taking experiment approaches and the fourth a computation approach to explore metabolic interaction. The experiment papers all involve the characterisation of fluxes. The first of these (Szecowka et al. 2013) does not directly but rather indirectly address photorespiration. It is discussed elsewhere in this article. The other two studies (Abadie et al. 2018; Abadie and Tcherkez 2019) used NMR analysis to characterize carbon fluxes in sunflower leaves. One demonstrated that the in vivo phosphoenolpyruvate carboxylase catalysed flux from bicarbonate to four-carbon acids is controlled by the mole fractions of CO₂ and oxygen and represents a major flux at high photorespiratory rates (Abadie and Tcherkez 2019). The other looked at the carbon allocation to major metabolites of the sunflower, namely glutamate, alanine, glycine, serine and chlorogenate and looked at their incorporation of ¹³C following isotopic labelling with ¹³CO₂ under different CO₂/oxygen conditions finding that the accumulation of ¹³C in these compounds is influenced by the gaseous conditions and thereby indicating the interconnection of photorespiration also with the metabolic pathways involved in their biosynthesis (Abadie et al. 2018). In the fourth paper, Huma et al. (2018) constructed a stoichiometric model of photorespiration and subjected it to elementary flux mode analysis – a technique which enumerates all the component minimal pathways of a network. In their study they delineated not only the classical photorespiratory pathway but also modes involving photorespiration coupled to mitochondrial metabolism and ATP production, the glutathione-ascorbate cycle and nitrate reduction. These findings directly mirrored many of the experimental findings described above, and they also provided a demonstration that photorespiration itself only impacts the assimilation quotient (CO₂-to-oxygen ratio) in instances associated with concomitant nitrate reduction.

IV. Past and Future of Photorespiration

Photorespiration has an evolutionary history of several billion years and similar to the Calvin-Benson cycle is a blend of archaeal, proteobacterial and cyanobacterial enzymes (Bauwe et al. 2012; Kern et al. 2015; Gütle et al. 2016; Hagemann et al. 2016).

A. Early Steps

The history of glyoxylate and related compounds, such as glyoxal, glycolaldehyde, glycolate and glycine, reaches back to abiotic times (Miller 1953; Menor-Salván 2018). Grounded on prebiotic reactions, first organic life dates back to 4.1–3.9 billion years ago, and it is thought that the last universal common ancestor of archaea, bacteria, and eukaryotes (LUCA) was an autotroph methanogen living in and on hot submarine hydrothermal vents. This organism likely used hydrogen as an energy source in combination with the THF-based chemistry of the acetyl-CoA (or Wood-Ljungdahl) pathway for CO₂ fixation (Cotton et al. 2018). From here, two different domains of life diverged, Archaea and Bacteria, followed by the evolution of eukaryotes from partnership between an Archaeon and a bacterial endosymbiont more than two billion years later (reviewed in Eme et al. 2017).

Complementing autotrophy, heterotrophic pathways evolved that, for example, used ribose moieties derived from RNA degradation. One of these pathways, designated the ‘pentose bisphosphate pathway’, operates in the archaeon Thermococcus kodakarensis to convert ribose into RuBP which is then carboxylated by a type III Rubisco to produce 3PGA (Aono et al. 2015). A related Rubisco-mediated but cyclic and autocatalytic ‘reductive hexulose phosphate pathway’ operates in in other archaea (Kono et al. 2017). This pathway takes up and releases equimolar amounts of CO₂ and formaldehyde, respectively, driving methane synthesis and/or the reductive acetyl-CoA pathway of CO₂ fixation. The authors speculate that the whole Calvin-Benson cycle may have originated from such an archaeal pathway. Actually, while the Calvin-Benson cycle by far predominates primary production in the extant biosphere, it is is just one, and the youngest in evolutionary terms, of six CO₂ fixation pathways found in nature (Fuchs 2011).

When the dual-photosystem bearing, oxygen-evolving cyanobacteria evolved is actively debated. It could have happened as early as 3.2 billion years ago as indicated by earliest traces of oxygenic photosynthesis; other offered dates span more than a billion years (reviewed in Schirrmeister et al. 2016). Concerning the how, it is broadly accepted that cyanobacteria acquired the ability for oxygenic photosynthesis by lateral gene transfer, PS I from green sulphur bacteria, PS II from anaerobic phototrophs such as purple bacteria, whereas the Rieske/cytochrome b complex was inherited vertically (reviewed in Fischer et al. 2016), resulting in the divergence of non-photosynthetic and photosynthetic cyanobacteria from anoxygenic ancestors at least 2.5–2.6 billion years ago (Soo et al. 2017).

These ancestral ‘oxyphotobacteria’ were the first to be challenged by high to super-saturating daytime oxygen tensions in locally oxic environments, particularly in microbial mats (reviewed in Dick et al. 2018; Hamilton 2019), triggering new metabolic processes and adaptations including massively increased rates of 2PG synthesis and its degradation by an ancient photorespiratory pathway. Reconstruction of this pathway revealed that cyanobacterial 2PG recycling typically comprises two partially redundant pathways, a photorespiratory pathway very similar to that of plants plus the bacterial glycerate pathway (Fig. 12.4; Eisenhut et al. 2008). Both pathways start by 2PG dephosphorylation and the oxidation of glycolate to glyoxylate by glycolate dehydrogenase and/or a GOX-like oxidase (Hackenberg et al. 2011; Hagemann et al. 2016). The glycerate pathway circumvents the glycine-to-serine conversion by directly converting glyoxylate into glycerate using tartronate-semialdehyde synthase and tartronate-semialdehyde reductase. Only few advanced cyanobacteria have a 3PGA-forming GLYK; most use 2PGA-forming glycerate kinases in combination with phosphoglyceromutase for the regeneration of 3PGA. Some cyanobacteria can also completely decompose glyoxylate to CO₂ via oxalate decarboxylase and formate dehydrogenase. If all three routes are inactivated, the mutant cyanobacteria can no longer grow in normal air and require elevated CO₂ to survive (Eisenhut et al. 2008). Most extant cyanobacteria have highly efficient CO₂ concentrating mechanisms (CCMs) to capture and concentrate CO₂ and hydrogencarbonate even at a very low concentration (Badger and Price 2003). It is remarkable that CCMs do not fully suppress 2PG synthesis – functional photorespiratory metabolism is still essential for photoautotrophic growth. It is also notable that even the relatively small genomes of all marine picocyanobacteria harbor almost all the genes necessary to express a plant-like 2PG metabolism as well as the glycerate pathway (Scanlan et al. 2009).

Fig. 12.4.

Most cyanobacteria recycle phosphoglycolate via two partially redundant pathways, a plant-like photorespiratory pathway (shown in light blue) and the bacterial glycerate pathway (orange route). Both pathways start with PGLP and glycolate dehydrogenase (GlcDH). The glycerate pathway circumvents the glycine-to-serine conversion by directly converting glyoxylate into glycerate using tartronate-semialdehyde synthase (TSS) and tartronate-semialdehyde reductase (TSR). Some cyanobacteria can also completely decompose glyoxylate to CO₂ (grey route) via oxalate decarboxylase (ODC) and formate dehydrogenase (FDH). Note this scheme is modified from Bauwe (2019)

After these rather local changes, the radiation of cyanobacterial photosynthesis enriched the atmosphere with oxygen and led to the Great Oxidation Event (GOE) about 2.3–2.4 billion years ago, when the oxygen content of the atmosphere rose from essentially anoxic 10⁻⁵ to about 10⁻² of its present atmospheric level (PAL). It may have been that increasing oxygen levels destroyed a methane greenhouse and triggered several major glaciations; most importantly, however, they enabled the evolution of aerobic heterotrophic life (see for example, Knoll and Nowak 2017). Overwhelming evidence suggests moderately oxic surface waters in the oceans then coexisted with anoxic deeper strata for a very long time, from 1800 to 800 million years ago (the ‘Boring Billion’), during which tight feedback from the oxidation of sulphide by anoxygenic photoautotrophs, nitrification and maybe a biospheric dependence on cyanobacterial N₂-fixation hindered further oxygen accumulation (reviewed in Dick et al. 2018). At the same period of time, low-nutrient pressures may have triggered essential biological innovations, particularly the establishment and radiation of the eukaryotic domain (Mukherjee et al. 2018).

B. Eukaryote Evolution in a Nutshell

The multi-step origin of eukaryotes lies within both the Archaea domain and the Proteobacteria (reviewed in Dacks et al. 2016). One of the suggested scenarios involves that a toxic rise in O₂ levels in ancient Archaean microenvironments, due to cyanobacterial photosynthesis, could have driven the establishment of an oxygen-tolerant archaeal lineage and its transformation into the first stem eukaryotes (Gross and Bhattacharya 2010). Molecular clock approaches based on the O₂-intensive process of sterol biosynthesis, which was likely transferred from bacteria to archaea by horizontal gene transfer (HGT), date this event in the time of GOE (Gold et al. 2017).

Whether the acquisition of mitochondria was an earlier or later event in eukaryogenesis is still debated (Roger et al. 2017). Here, it is interesting that mitochondria are not absolutely essential for the viability of a eukaryotic cell (Karnkowska et al. 2016), which favours the view that mitochondrial endosymbiosis at least was not the first event in eukaryogenesis. Presently, a widely advocated hypothesis suggests that the closest relative to mitochondria-containing eukaryotes on the host side was a hydrogen-consuming anaerobic phagotrophic Archaeon of the Asgard supergroup, which around 1.5 billion years ago engulfed a proteobacterium that eventually became the mitochondrion (Eme et al. 2017; Martijn et al. 2018). There is evidence that the pre-mitochondrial endosymbiont diverged from either the Rickettsiales order of the Alphaproteobacteria or from a proteobacterial lineage that branched off before the divergence of Alphaproteobacteria (Martijn et al. 2018). By contrast, the single-membrane-bounded peroxisome did not evolve by endosymbiosis but can be generated de novo by the fusion of endoplasmic reticulum (ER)-derived buds (van der Zand and Tabak 2013) or as hybrids from ER- and mitochondria-derived pre-peroxisomes (Sugiura et al. 2017; Kao et al. 2018). Peroxisomes house diverse functions and with some exceptions, where they have been lost, occur in nearly all eukaryotic cells.

C. Plant Photorespiration: A Blend of Archaeal and Bacterial Enzymes

All Archaeplastida trace their origin to the so-called primary endosymbiontic event during the mid-Proterozoic, when a biflagellate phagotrophic eukaryote engulfed a photosynthetic cyanobacterium similar to the extant freshwater cyanobacterium Gloeomargarita lithophora to become the primary chloroplast (Ponce-Toledo et al. 2017; review in Nowack and Weber 2018). This was the first major split in eukaryote evolution, during which photosynthesis and the basic frame of the photorespiratory pathway were conveyed to algae and land plants (Eisenhut et al. 2008). The extensive radiation of Rhodophyta and Chlorophyta then occurred between 1600 and 1000 and 1000–542 million years ago, respectively (Sánchez-Baracaldo et al. 2017). Recent data identified the charophytic alga Chara braunii at the root of land plants (Nishiyama et al. 2018). While cyanobacteria now are an established prokaryotic model of the function and evolution of photorespiration in embryophytes (Orf et al. 2016), there are some gaps in our knowledge though. To mention just one example, cyanobacterial and plant PGLP belong to different haloacid dehalogenase families, and their evolutionary relationship is still uncertain (Rai et al. 2018). Altogether, it appears that during the evolution of algae and land plants multiple losses and replacements occurred, which resulted in a reticulate provenance of photorespiratory enzymes with different origins in different cellular compartments (Kern et al. 2013).

When land plants appeared on Earth about 500 million years ago (Morris et al. 2018), oxygen levels had increased from likely approximately 0.2–0.3% at the time of the endosymbiotic event leading to chloroplasts to approximately the levels we have today; however, CO₂ was still 15-fold higher than in our present atmosphere (Hetherington and Raven 2005). Massive photosynthetic activity in the Carboniferous period (360–300 million years ago) then resulted in an intermittent drop in CO₂ levels and an intermittent further rise in oxygen levels. During the past 25 million years, CO₂ levels were generally lower than today.

In addition to atmospheric CO₂ levels, temperature was another important factor in some areas because the specificity of Rubisco for CO₂ over oxygen is worse at high temperatures and the solubility of oxygen declines more slowly than that of CO₂. Approximately 30 million years ago, as an adaptation to lower CO₂ levels and higher temperatures, a number of land plants independently from each other evolved CCMs that are known as C₄ photosynthesis (Christin et al. 2008). This pathway is based on a pre-fixation of CO₂ in the mesophyll by phosphoenolpyruvate carboxylase to first make a four-carbon (C₄) compound. The enzyme efficiently captures CO₂ even at a very low concentration, creating a large gradient in CO₂ concentration between the inside of the leaf and the outside environment. The C₄ compound then moves to the Rubisco-containing bundle sheath cells, where the CO₂ is released by a C₄ acid decarboxylase. Through this mechanism, Rubisco operates at greatly elevated CO₂ levels and 2PG synthesis is low though not entirely absent. Despite these advantages, C₄ photosynthesis is not competitive in all climates and far most land plants use the C₃ photosynthetic pathway (Sage et al. 2018).

Paradoxically, photorespiration not only provided pressure towards the evolution of C₄ photosynthesis but also triggered this process by inventing the first though not yet very efficient plant CCM (Bauwe 2011). This was worked out by the study of C₃-C₄ intermediate plants. These predecessors of C₄ plants produce glycine in all photosynthetic cells, but, due to a restriction of the GCS to the bundle sheath, the photorespiratory glycine is decarboxylated only in the vein-surrounding bundle sheath cells (Rawsthorne 1992; Schulze et al. 2013; Keerberg et al. 2014). The resulting higher concentration of photorespired CO₂ in the bundle sheath and the need to return the surplus photorespiratory ammonia nitrogen back to the mesophyll prepared the path for C₄ plant evolution (Mallmann et al. 2014).

D. Crop Improvement

Rubisco is responsible for nearly all biological CO₂ fixation on Earth, resulting in a terrestrial gross primary productivity (true photosynthesis minus photorespiration) of about 120 billion tons carbon year⁻¹ on land plus a similar productivity of the oceans (Field et al. 1998; Geider et al. 2001). In molecular terms, about every third molecule of RuBP becomes oxygenated instead of carboxylated in a C₃ plant growing under temperate conditions in the present atmosphere (Fig. 12.1; Bauwe et al. 2012). Photorespiration rates accordingly can approach 50% of the net-photosynthesis in most land plants and be even higher in warm and dry environments (Sage and Kubien 2007; Bauwe 2019; Slattery and Ort 2019). Thus, on a global scale, an estimated ~30 billion tons year⁻¹ carbon re-liberated from leaf photorespiration and day respiration need to be added to the above numbers (Tcherkez 2013). Additionally, the energy requirements for the recycling of 2PG into RuBP and photorespiratory ammonia into glutamate nitrogen can represent more than one third of the total energetic costs of CO₂ fixation (Walker et al. 2016). Reducing these CO₂ and energetic losses has been a sensible target for molecular breeding for decades but there are no trivial solutions and any such attempt must consider how photorespiration is embedded into photosynthetic and mainline metabolism.

Although real-life photosynthetic carbon metabolism involves additional ancillary metabolism (Huma et al. 2018; Tcherkez and Limami 2019), at first sight, the correlation between photorespiration and (net-) photosynthesis in a C₃ plant is as straightforward as it is simple. The correlation is positive (!) as demonstrated in many studies including a comparison of Arabidopsis ecotypes (Tomeo and Rosenthal 2018) and large-scale field studies with various wheat and soybean genotypes, in which the high-productive genotypes showed high rates of photosynthesis along with high rates of photorespiration (Aliyev 2012). The rash conclusion is that the breeders’ primary focus on the physiological level should be net-photosynthesis irrespective of the level of photorespiration, but this view would miss the potential opportunities given by enzyme and metabolic engineering.

Concerning better crops for the future, one focus is on the improvement of Rubisco‘s catalytic efficiency, including a better ratio of carboxylation versus oxidation of RuBP, by directed evolution and introduction of such an enzyme into the chloroplast of C₃ crops (Carmo-Silva et al. 2015; Sharwood 2017; Flamholz et al. 2019; Zhou and Whitney 2019). A second group of approaches aims at the 2PG production by elevating the CO₂ concentration around Rubisco via the establishment of C₄- and cyanobacteria-like CCMs in C₃ plants, particularly rice (Wang et al. 2016; Long et al. 2018). Third, artificial bypasses to the photorespiratory pathway could reduce or possibly fully avoid the release of photorespiratory ammonia and the resulting high energetic costs of refixation (Betti et al. 2016; Bar-Even 2018). To this end, several complex artificial pathways have been designed that could assimilate CO₂ without photorespiration (Schwander et al. 2016; Bar-Even 2018) or convert glycolate in a CO₂-fixing 3-hydroxypropionate cycle (Shih et al. 2014). The most recent addition involves two newly engineered enzymes to convert glycolate to glycolaldehyde, followed by the condensation of glycolaldehyde and phosphoglyceraldehyde (via aldolase) to form arabinose 5-phosphate and then (via additional enzymes) RuBP (Trudeau et al. 2018). As yet, this latter approach has been tested in vitro while tests in planta are still up in the air. An easier approach is the conversion of glyoxylate to glycerate in the chloroplast, as it happens in bacteria including cyanobacteria (Eisenhut et al. 2006), or in the peroxisome. In either case, glyoxylate would be converted to glycerate without prior conversion to glycine. Both variants were tested by using enzymes of the bacterial glycerate pathway but with inconsistent results. Overexpression of the bacterial glycerate pathway in the chloroplast of several plant species consistently improved photosynthesis and growth under controlled conditions (Kebeish et al. 2007; Bai et al. 2011; Dalal et al. 2015) and in the field (South et al. 2019), whereas plant growth was impaired following peroxisomal overexpression (Carvalho et al. 2011; Chen et al. 2019). Finally, maybe inspired by the photorespiration driven CCM operating in C₃-C₄ plants, several groups tried to achieve full oxidation of glycolate to CO₂ within the chloroplast. Whilst this approach is not fully comprehensible for theoretical reasons (for example Xin et al. 2015), the generated transgenic plants nevertheless displayed improved photosynthesis parameters and better growth (Maier et al. 2012; Shen et al. 2019; South et al. 2019). The chloroplastidal overexpression of only the E. coli (Nölke et al. 2014; Chen et al. 2019) or cyanobacterial (Ahmad et al. 2016; Bilal et al. 2019) glycolate dehydrogenase or related enzymes however produced similar growth effects. The functionality of the intended artificial bypasses actually was not directly examined at the molecular level in any of these studies and, altogether, the molecular mechanism of how the higher photosynthetic efficiency was achieved in these lines is not yet known. Potential benefits and drawbacks of the above bypasses were critically tested by using model-based approaches (Xin et al. 2015; Basler et al. 2016). These authors stress the importance of additional factors such as the intra-cellular diffusion of CO₂ and the interaction of photorespiration with other metabolic pathways.

Indeed, exploitation of regulatory feedback from the photorespiratory pathway to the operation of the Calvin-Benson cycle is another way to enhance net photosynthesis. To some extent, this strategy is based on the well-established and already mentioned fact that any impairment of photorespiratory carbon flow results in an impairment of photosynthesis (discussed in Timm et al. 2016). Mechanistically, the underlying regulation could involve photorespiratory metabolites such as 2PG, glycolate, glyoxylate, and glycine. Glycolate itself is not toxic to Chlamydomonas (Taubert et al. 2019) but easily convertible to glyoxylate and glycine, for which inhibitory effects were reported (Chastain and Ogren 1989; Eisenhut et al. 2007). Indeed, overexpression of GCS proteins and other enzymes of the photorespiratory pathway produced plants with improved photosynthetic efficiency and biomass production (Timm et al. 2012a; Timm et al. 2015; Simkin et al. 2017; Lopez-Calcagno et al. 2018). It must be stressed again though that the molecular mechanism of this inter-organellar crosstalk is not known. Better investigated is the regulation of several Calvin-Benson cycle enzymes by the inhibitor 2PG, modulating RuBP regeneration and starch synthesis (Flügel et al. 2017).

During the past 60 years, global CO₂ concentration as continuously measured since 1958 (Keeling and Rakestraw 1960) has increased from ~0.032% to a present level of ~0.041%. This change has decreased the photorespiration/photosynthesis ratio by about 25% worldwide over the twentieth century (Ehlers et al. 2015) and, as the CO₂ concentration continues to increase in the future, photorespiration will continue to fall. The higher efficiency of C₃ plants under these conditions could be levelled to some extent by global warming (discussed in Dusenge et al. 2019), which by and large promotes C₄ more than C₃ plants, but might affect competition between C₃ and C₄ plants and alter the species composition and the diversity in many ecosystems (Ehleringer et al. 1997). Some researchers believe that higher atmospheric CO₂ levels will increase crop yields (for example Walker et al. 2016); however, global climate is difficult to predict and local changes to temperature , rainfall and other determinant factors for agricultural productivity could more than offset any productivity gains at a given location.

V. Outlook

Photorespiration is an important high-flux bearing metabolic process that enables photosynthetic CO₂ fixation based on photochemical water-splitting and, thereby, is a factor that co-determines crop yields and influences the global carbon dynamics. Due to its evolutionary history, plant photorespiration is a blend of archaeal and bacterial enzymes operating in different subcellular compartments. The pathway hence requires transmembrane metabolite passages. As yet, not all involved transporter proteins are known, but several were identified in recent years, such as PLGG1 and BASS6 and the indirectly involved BOU. The detailed function of these and further proteins, such as the ER-ANT1 transporter, remains to be explored.

Plant photorespiration is much more complex than thought still one decade ago, interacting with other day-time metabolism in a variety of ways. For example, interaction with the Calvin-Benson cycle goes much beyond the mere recycling of 2PG and involves regulatory interdependencies operating on multiple levels. Chloroplastidal levels of 2PG but maybe also glycolate and glyoxylate and other metabolites are seemingly central for balancing this web. Important examples of other newly discovered but not yet fully understood interactions concern starch synthesis, mitochondrial respiration, sulphur metabolism, nitrogen assimilation and cyclic electron transport. Future research will likely focus on the post-translational and other biochemical regulation of this network.

Other exciting research of recent years provided an unexpected view of the role played by photorespiration during the evolution of C₄ plants. It appears that this process began with a photorespiration-based CCM, using glycine as a vehicle for freshly assimilated CO₂, followed by the establishment of the C₄ cycle CCM from enzymes that initially balanced the intercellular nitrogen status. Related work explained how the intercellular division of photosynthetic-photorespiratory labour integrates with cellular one-carbon metabolism of C₄ plants.

Due to the ever growing need for high-yield crops, last years have shown an impressive revival of photorespiration research. Particularly, a number of approaches have impressively demonstrated the high potential for better crop yields of optimising the photorespiratory carbon and nitrogen flow. Not all of this work was initially designed to achieve better plant growth but rather served to better understand how photorespiration actually works and is embedded into whole plant metabolism. This is changing. In light of the achieved progress, new and more rational strategies for crop improvement are being designed and tested, which could be one of the most important avenues of photorespiration research for the next decade.

Acknowledgements

Our research on photorespiration was generously supported by the Deutsche Forschungsgemeinschaft (Research Unit FOR 1186 Promics, BA 1177/12 and FE 552/10) and inspired by the collaboration and discussions with many colleagues of the Promics network and beyond.