2.3.1  Evidence for photorespiration

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Figure 2.15 Illuminated (detached) tobacco leaves were held in a closed system of recirculating gas (initially 100 µL L-1 CO2 and 21% O2) and allowed to equilibrate with ambient CO2, thereby obtaining a CO2 compensation point. O2 levels were varied between 2% and 47% in subsequent experiments. The CO2 compensation point at equilibrium showed a linear dependence on surrounding O2 concentration. Data from two leaves have been combined (Based on Krotkov 1963)

The most obvious demonstration that a net release of respiratory CO2 occurs in light is the CO2 compensation point. 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 (Egle and Schenk 1953; Tregunna et al. 1966; Zelitch 1966). Response to O2 has some important implications and in low concentrations (1–2% O2) the CO2 compensation point is near zero (Figure 2.15) while net photosynthesis is increased by low O2 (Tregunna et al. 1966).

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; Forrester et al. 1966) and raised a very perplexing question as to whether these species even respired in light. However, we now know that C4 photosynthesis is responsible (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 2.16 Photosynthesising leaves show a post-illumination burst of CO2 which varies in strength according to surrounding O2 concentration. This positive response to O2 was observed at c. 105 umol quanta m-2 s-1 and is functionally linked to O2 effects on the CO2 compensation point (Figure 2.15) 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 (Figure 2.16). This ‘post-illumination CO2 burst’ was studied extensively during the early 1960s by Gleb Krotkov and colleagues at Queens University (Kingston, Ontario) (Krotkov 1963). The intensity of this burst was found to be sensitive to O2 (Figure 2.16) and was closely related to photon irradiance during the preceding period of photosynthesis (Figure 2.17). 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 also abolished in low O2 (2% O2 in Figure 2.16; see also Tregunna et al. 1966). Indeed, a competitive inhibition by O2 on CO2 assimilation was suspected and was subsequently proved to be particularly relevant in defining Rubisco’s properties. Never-theless, for many years a biochemical explanation for inter-action between these two gases remained elusive.

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Figure 2.17 Photosynthesising leaves (in 21% O2) show a post-illumination burst of CO2 which varies in strength according to the intensity of preceding illumination shown here to range from 35 to 700 pmol quanta m-2 s-1 on each graph. (Original data in 'foot candles' have been converted to photon irradiance.) A large outburst occurred within the first minute with a second weaker emission of longer duration. Steady rates of CO2 production were reached after c. 6 min. (Based on Krotkov 1963)

Significant progress came when Ludwig and Krotkov (1967) 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. 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. In essence 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. Indeed 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 con-centration (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. Clearly 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.

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