2.3.3  Localisation of photorespiration

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The 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) and Tolbert (1963) 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. Indeed 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 devel-opment 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 photo-respiration 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.

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Figure 2.18 Photosynthetic carbon reduction (PCR) and photosynthetic carbon oxidation (PCO) pathways in green leaves are closely integrated. Rubisco in chloroplasts is initially responsible for both carboxylation and oxygenation of RuBP, and the balance between RuBP carboxylase (A) and RuBP oxygenase (B) reactions dictates the comparative rates of these two pathways. That poise in turn affects the net uptake of CO2 per RuBP consumed and the energy input as ATP or NADPH that is required for net uptake of CO2. Energy (ATP) and reducing power (NADPH) derived from photosynthetic membranes are used to drive both cycles.Three ATP and two ADPH are consumed in regeneration of each RuBP via the PCR cycle; whereas 3.5 ATP and two NADPH are required for each oxygenation via the PCO pathway. One CO2 is produced by photorespiration for every two oxygenations. (Based on Berry and Bjorkman 1980)

A simplified scheme for the PCR cycle and photosynthetic carbon oxidation (PCO) pathways in Figure 2.18 represents the synthesis of almost 70 years of research effort, and integrates the metabolism of P-glycolate with the PCR cycle. Specialised reactions within three classes of organelles in leaf cells are required, namely chloroplasts, microbodies and mitochondria. Their close proximity in leaf cells (Figure 2.11) plus specific membrane transporters (Secton 2.1.8) facilitate metabolite exchange. Oxygenase activity by Rubisco results in formation of phosphoglycolate (within chloroplasts) which then enters a PCO cycle, and is responsible for loss of some of the CO2 just fixed in photosynthesis. During photorespiration, O2 is consumed in converting glycolate to glyoxylate (within peroxisomes), while further CO2 is released during subsequent condensation of glycine to serine (within mitochondria). 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.

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 (Km = 12 µM) compared with a much lower affinity for O2 (Km = 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! Aided by an ordered reaction, CO2 assimilation prevails, and a net fixation of carbon is an obvious outcome.

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. This ammonia would be extremely toxic if it were not to be reassimilated by either cytosolic or chloroplastic glutamine synthetase. Indeed very effective herbicides which block glutamine synthetase have been developed (phosphinothricin or glufosinate) and when these are applied to actively growing plants they are killed by their photorespiratory ammonia release (herbicides are discussed further in Chapter 20).

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