(a) Historical evidence for photorespiration

The first line of evidence for photorespiration came from the different compensation points of C3 and C4 plants. When air is recirculated over an illuminated leaf in a closed system, photosynthesis will reduce CO₂ concentration to a low level where fixation of CO₂ by photosynthesis is just offset by release from respiration. For many C₃ plants this compensation point is around 50 µL L^–1 but is markedly affected by oxygen, photon irradiance and leaf temperature (Tregunna et al. 1966; Zelitch 1966). In low concentrations (1–2% O₂) the CO₂ compensation point of C3 plants is near zero. Significantly, early researchers in this area had already noted that some tropical grass species appeared to have a compensation point at or close to zero CO₂, even in normal air (20% O₂). This was first reported for corn (Zea mays) (Meidner 1962) and raised a very perplexing question as to whether these species even respired in light. However, we now know that C₄ photosynthesis is responsible for the low evolution of CO₂ (Section 2.2) and that C₄ plants have a CO₂ concentrating mechanism that forestalls photorespiration, resulting in a CO₂ compensation point close to zero.

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Figure 2.12. Photosynthesising leaves show a post-illumination burst of CO₂ which varies in strength according to surrounding O₂ concentration. This positive response to O₂ was found at 105 µmol quanta m^-2 s^-1 and is functionally linked to O₂ effects on the CO₂ compensation point as measured under steady-state conditions. (Based on Krotkov 1963)

A second line of evidence for leaf respiration in light was provided by a transient increase in release of CO₂ when leaves are transferred from light to dark. This ‘post-illumination CO₂ burst’ was studied extensively during the early 1960s by Gleb Krotkov and colleagues at Queens University (Kingston, Ontario). The intensity of this burst increased with the photon irradiance during the preceding period of photosynthesis. Understandably, the Queens group regarded this post-illumination burst as a ‘remnant’ of respiratory processes in light, and coined the term ‘photorespiration’. A functional link with the CO₂ compensation point was inferred, because the burst was abolished in low O₂ (Figure 2.12). A competitive inhibition by O₂ on CO₂ assimilation was suspected and was subsequently proved to be particularly relevant in defining Rubisco’s properties. Nevertheless, for many years a biochemical explanation for interaction between these two gases remained elusive.

(b) Biochemistry

Significant progress came when Ludwig and Krotkov designed an open gas exchange system in which ¹⁴CO₂ was used to separate the fixing (photosynthetic) and evolving (respiratory) fluxes of CO₂ for an illuminated leaf (Ludwig and Canvin 1971). Results using this steady-rate labelling technique were particularly revealing and provided the first direct evidence that respiratory processes in light were qualitatively different from those in darkness. They were able to show that CO₂ evolved during normal high rates of photosynthesis by an attached sunflower leaf was derived from currently fixed carbon. The specific activity of evolved CO₂ (ratio of ¹⁴C to ¹²C) was essentially the same as that of the CO₂ being fixed, indicating that photorespiratory substrates were closely related to the initial products of fixation. Ludwig and Krotkov concluded that ¹⁴CO₂ supplied to a photosynthesising leaf was being re-evolved within 28–45 s! Furthermore the rate of CO₂ evolution in light was as much as three times the rate in darkness, and while early fixed products of photosynthesis (intermediates of the PCR cycle) were respired in light, this was not the case in darkness.

The radiolabelling method of Ludwig and Krotkov had, for the first time, provided measurements of what could be regarded as a true estimate of light-driven respiration which was not complicated by transient effects (as the post-illumination burst had been), or by changes in CO₂ concentration (as was the case for measurements in closed gas exchange systems or in CO₂-free air) or by difficulties associated with detached organs. Ludwig and Canvin (1971) subsequently concluded that processes underlying photorespiration re-evolved 25% of the CO₂ which was being fixed concurrently by photosynthesis. Such a rate of CO₂ loss was not a trivial process so a biochemical basis for its operation had to be established, and particularly when photorespiration seemed to be quite different from known mechanisms of dark (mitochondrial) respiration.

The search for the substrates of ‘photorespiration’ occupied many laboratories worldwide for many years. Much work centred on synthesis and oxidation of the two-carbon acid glycolate because as early as 1920 Warburg had reported that CO₂ fixation by illuminated Chlorella was inhibited by O₂, and under these conditions the alga excreted massive amounts of glycolic acid (Warburg and Krippahl 1960). Numerous reports on the nature of the ¹⁴C-labelled products of photosynthesis showed that glycolate was a prominent early-labelled product. A very wide variety of research with algae and leaves of many higher plants established two significant features of glycolate synthesis: formation was enhanced in either low CO₂ or high O₂. Both of these features had been predicted from Ludwig’s physiological gas exchange work and eventually proved a key to understanding the biochemistry of photorespiration.

(c) Source of glycolate production

The photosynthetic carbon reduction (PCR) cycle for CO₂ fixation (Section 2.1) involves an initial carboxylation of ribulose-1,5-bisphosphate (RuBP) to form 3-PGA, but makes no provision for glycolate synthesis. However, Wang and Waygood (1962) had described the ‘glycolate pathway’, namely a series of reactions in which glycolate is oxidised to glyoxylate and aminated, first to form glycine and subsequently the three-carbon amino acid serine. The intracellular location of this pathway in leaves was established in a series of elegant studies by Tolbert and his colleagues who also established that leaf microbodies (peroxisomes) were responsible for glycolate oxidation and the synthesis of glycine. Kisaki and Tolbert (1969) suggested that the yield of CO₂ from the condensation of two molecules of glycine to form serine could account for the CO₂ evolved in photorespiration. This idea was incorporated in later formulations of the pathway.

What remained elusive was the source of photosynthetically produced glycolate. Many studies had suggested that the sugar bisphosphates of the PCR cycle could yield a two-carbon fragment which, on the basis of short-term ¹⁴CO₂ fixation, would have its two carbon atoms uniformly labelled (if the two carbons were to be derived directly from 3-PGA this would not be the case as PGA was asymmetrically labelled in the carboxyl group). The mechanism was likened to the release of the active ‘glycolaldehyde’ transferred in the thiamine pyrophosphate (TPP)-linked transketolase-catalysed reactions of the PCR cycle. In some cases significant glycolate synthesis from the sugar bisphosphate intermediates of the cycle were demonstrated in vitro; however, the rates were typically too low to constitute a viable mechanism for glycolate synthesis in vivo.

A more dynamic approach to carbon fixation was needed to resolve this impasse. In particular, the biochemical fate of early products would have to be traced, and using a development of the open gas exchange system at Queens, Atkins et al. (1971) supplied ¹⁴CO₂ in pulse–chase experiments to sunflower leaf tissue under conditions in which photorespiration was operating at high rates (21% O₂) or in which it was absent (1% O₂). A series of kinetic experiments showed that synthesis of ¹⁴C-glycine and ¹⁴C-serine was inhibited in low O₂ and that the ¹⁴C precursor for their synthesis was derived from sugar bisphosphates of the PCR cycle, especially RuBP. Indeed RuBP was the obvious source of glycine carbon atoms and the kinetics of glycine turnover closely matched those of RuBP. As these authors concluded, ‘we can no longer view this (glycolate) pathway as an adjunct to the Calvin cycle but must incorporate it completely into the carbon fixation scheme for photosynthesis’ (Atkins et al. 1971).

The question was finally and most elegantly resolved by Ogren and Bowes (1971) who demonstrated that the carboxylating enzyme of the PCR cycle, RuBP carboxylase, was both an oxygenase and a carboxylase! During normal photosynthesis in air, this enzyme thus catalysed formation of both P-glycolate (the precursor of glycolate) and 3-PGA from the oxygenation of RuBP as well as two molecules of PGA from carboxylation of RuBP. In effect, CO₂ and O₂ compete with each other for the same active sites for this oxygenation/carboxylation of RuBP, at last providing a biochemical mechanism which had confused and perplexed photosynthesis researchers since the 1920s. This primary carboxylating enzyme of the PCR cycle, which had hitherto rejoiced in a variety of names (carboxydismutase, fraction 1 protein, RuDP carboxylase and RuBP carboxylase), was renamed Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) to reflect its dual activity.

A scheme for the PCR cycle and photosynthetic carbon oxidation (PCO) pathways then represents the synthesis of almost 70 years of research effort, and integrates the metabolism of P-glycolate with the PCR cycle. This is shown in the following section. 

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Figure 2.13. The photorespiratory carbon oxidation (PCO) cycle involves movement of metabolites between chloroplasts, peroxisomes and mitochondria. (Original drawing courtesy lan Woodrow)

A scheme for photorespiration involving the photosynthetic carbon reduction (PCR) cycle and the photosynthetic carbon oxidation (PCO) pathways represents the synthesis of almost 70 years of research. It integrates the metabolism of P-glycolate with the PCR cycle. This scheme is shown in Figure 2.13.

Specialised reactions within three classes of organelles in leaf cells are required, namely chloroplasts, peroxisomes (originally called microbodies) and mitochondria. Their close proximity in leaf cells (Figure 2.14 below) plus specific membrane transporters facilitate the exchange of metabolites.

Within choroplasts, oxygenase activity by Rubisco results in formation of phosphoglycolate which then enters a PCO cycle, and is responsible for loss of some of the CO₂ just fixed in photosynthesis.

Within peroxisomes, O₂ is consumed in converting glycolate to glyoxylate, and aminated to form glycine.

Within mitochondria, CO₂ is released during conversion of glycine to serine. Subsequently, serine is recovered by peroxisomes where it is further metabolised, re-entering the PCR cycle of chloroplasts as glycerate. About 75% of carbon skeletons channelled into photorespiration are eventually recovered as carbohydrate.

Transport of glycerate and glycolate across the inner membrane of chloroplasts may involve separate translocators as shown in Figure 2.13, or it may involve a single translocator that exchanges two glycolate molecules for one molecule of glycerate. Transport of metabolites across the peroxisomal membrane most likely occurs through unspecific channel proteins, similar to those in the outer membranes of mitochondria and chloroplasts. These outer membranes are not included in this diagram. Mitochondria take up two molecules of glycine and release one molecule of serine. A specific translocator most probably mediates the exchange of these amino acids.

Not only does the photosynthetic oxidation pathway consume O₂ and release CO₂ but ammonia is also produced by mitochondria during synthesis of serine from glycine (Figure 2.13). This ammonia would be extremely toxic if it were not metabolised by either cytosolic or chloroplastic glutamine synthetase. A very effective herbicide that blocks glutamine synthetase has been developed (phosphinothricin, also known as glufosinate or Basta), and when it is applied to (or expressed in) actively growing plants they are killed by their photorespiratory ammonia release.

Fig 2.11.jpg

Figure 2.14. A transmission electron micrograph showing close juxtaposition of chloroplast (C), mitochondrion (M) and peroxisome (P) in a mesophyll cell of an immature leaf of bean (Phaseolus vulgaris). This group of organelles is held within a granular cytoplasmic matrix adjacent to a cell wall (CW) and includes a partial view of a small vacuole (V). Scale bar = 1 µm (Electron micrograph courtesy Stuart Craig and Celia Miller)

In summary, participation of photorespiration in leaf gas exchange, and thus dry matter accumulation by plants, reflects kinetic properties of Rubisco, and in particular a relatively high affinity for CO₂ (K_m = 12 µM) compared with a much lower affinity for O₂ (K_m = 250 µM). That contrast in affinity is, however, somewhat offset by the relative abundance of the two gases at catalytic sites of the enzyme where the ratio of O₂:CO₂ partial pressures approaches 1000:1! Thus, CO₂ assimilation always prevails over CO₂ loss in photorespiration. 

At around the same time as the nature of photorespiration was becoming clearer Hatch and Slack (1966) demonstrated that in tropical grasses (initially sugar cane) the first-formed products of photosynthetic CO₂ fixation were the four-carbon acids oxalacetate, malate and aspartate, rather than the 3-PGA formed in the PCR cycle. Furthermore, the carboxylation reaction involved PEP carboxylase and carbon was subsequently transferred to PCR cycle intermediates. As noted earlier (Section 2.2) C₄ plants show no apparent CO₂ release in light. The explanation lies in their anatomy and multiple carboxylation reactions rather than in the absence of the pathway of photorespiration. Bundle sheath cells are equipped with a CO₂-concentrating mechanism that favours carboxylation over oxygenation reactions due to increased partial pressure of CO₂, while photorespiratory release of CO₂ is further prevented through the activity of PEP carboxylase which refixes any respired CO₂ formed from the oxygenase function of Rubisco.

Unicellular green algae also posed a problem for the simple extrapolation of early models for photorespiratory metabolism in C₃ leaves. Although organisms such as Chlorella had been used to establish the PCR cycle, and indeed provided much early evidence for effects of O₂ on photosynthesis and formation of glycolate in light, they also appeared to lack CO₂ evolution in light (Lloyd et al. 1977). In this case the explanation lies in a CO₂-concentrating mechanism which effectively increases the internal pool of inorganic carbon (CO₂ and HCO₃^–) thereby favouring the carboxylase function of Rubisco over its oxygenase function.

Such a substantial loss of carbon concurrent with CO₂ fixation raises the question of whether eliminating or minimising photorespiration in C₃ plants could enhance their yield, and specifically that of major crop plants such as rice, wheat, grain legumes, oil seeds and trees, none of which are C₄ species. Faced with an expanding world human population and an increasing demand for food and animal feed, enhanced agricultural productivity is a global necessity. In its most obvious form a scenario which alters or removes the oxygenase function of Rubisco could achieve such a goal. In an early review of the process of photorespiration in plants, Ogren (1984) noted that ‘the sequence of reactions constituting the photorespiratory pathway in C₃ plants appears to be firmly established’ and he went on to suggest that, although reducing the loss of fixed carbon as CO₂ in the process may be a valid goal to improve the yield of crop plants, it is not clear whether or not this can be achieved by specific changes to the kinetic and catalytic properties of Rubisco alone.

Photorespiration may be loosely considered as a wasteful process because previously fixed carbon is lost and energy is dissipated. Ideal destinations for photoassimilates include synthetic pathways leading to fixed biomass and respiratory pathways for re-release of fixed energy in a controlled sequence of reactions leading to ATP and NAD(P)H for use in other synthetic events.

However, situations do exist where energy dissipation via photorespiration can be beneficial. For example, photo-oxidative damage can be alleviated in shade-adapted plants that experience strong irradiance if photorespiratory processes are allowed to proceed. Depriving such plants of an external oxygen supply, and hence preventing photosynthetic carbon oxidation, will exacerbate chloroplast lesions due to strong irradiance. Photosynthetic variants which obviate photo-respiratory loss, and most notably C₄ plants, integrate structure and function in a way that forestalls photo-oxidative damage and leads to their outstanding performance under warm conditions. Environmental factors that constitute selection pressure for such photosynthetic adaptation are reviewed by Sage et al (2012). 

Atkins CA, Canvin DT, Foch H (1971) Intermediary metabolism of photosynthesis in relation to carbon dioxide evolution in sunflower. In MD Hatch, CB Osmond, RO Slatyer, eds, Photosynthesis and Photorespiration. John Wiley, New York, pp 497-505

Hatch MD, Slack CR (1966) Photosynthesis by sugarcane leaves. A new carboxylation reaction and the pathway of sugar formation. Biochem J 101: 103-111

Kisaki T, Tolbert NE (1969) Glycolate and glyoxylate metabolism by isolated peroxisomes and chloroplasts. Plant Physiol 44: 242-250

Lloyd ND, Canvin DT, Culver DA (1977) Photosynthesis and photorespiration in algae. Plant Physiol 70: 1637-1640

Ludwig LJ, Canvin DT (1971) The rate of photorespiration during photosynthesis and the relationship of the substrate of light respiration to the products of photosynthesis in sunflower leaves. Plant Physiol 48: 712-719

Meidner HA (1962) The minimum intercellular-space CO₂ concentration of maize leaves and its influence on stomatal movements. J Exp Bot 13: 284-293

Ogren WL (1984) Photorespiration: pathways, regulation and modification. Annu Rev Plant Physiol 35: 415-442

Ogren WL, Bowes G (1971) Ribulose diphosphate carboxylase regulates soybean photosynthesis. Nature New Biol 230: 159-160

Sage RF, Sage TL, Kocacinar F (2012) Photorespiration and the evolution of C₄ photosynthesis. Annu Rev Plant Biol 63: 19-47.

Tregunna EB, Krotkov G, Nelson CD (1966) Effect of oxygen on the rate of photorespiration in detached tobacco leaves. Physiol Plant 19: 723-733

Warburg O, Krippahl G (1960) Glykolsaurebildung in Chlorella. Z Naturforsch 15b: 197-199

Wang D, Waygood ER (1962) Carbon metabolism of ¹⁴CO₂ labelled amino acids in wheat leaves. I. A pathway of glyoxylate-serine metabolism. Plant Physiol 37: 826-832

Zelitch I (1966) Increased rate of net photosynthetic carbon dioxide uptake caused by the inhibition of glycolate oxidase. Plant Physiol 41: 1623-1631