The cornerstone of autotrophy, the CO2-fixing enzyme, D-ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco), is hamstrung by slow catalysis and confusion between CO2 and O2 as substrates, an ''abominably perplexing'' puzzle, in Darwin's parlance. Here we argue that these characteristics stem from difficulty in binding the featureless CO2 molecule, which forces specificity for the gaseous substrate to be determined largely or completely in the transition state. We hypothesize that natural selection for greater CO2͞O2 specificity, in response to reducing atmospheric CO2:O2 ratios, has resulted in a transition state for CO2 addition in which the CO2 moiety closely resembles a carboxylate group. This maximizes the structural difference between the transition states for carboxylation and the competing oxygenation, allowing better differentiation between them. However, increasing structural similarity between the carboxylation transition state and its carboxyketone product exposes the carboxyketone to the strong binding required to stabilize the transition state and causes the carboxyketone intermediate to bind so tightly that its cleavage to products is slowed. We assert that all Rubiscos may be nearly perfectly adapted to the differing CO2, O2, and thermal conditions in their subcellular environments, optimizing this compromise between CO2͞O2 specificity and the maximum rate of catalytic turnover. Our hypothesis explains the feeble rate enhancement displayed by Rubisco in processing the exogenously supplied carboxyketone intermediate, compared with its nonenzymatic hydrolysis, and the positive correlation between CO2͞O2 specificity and 12 C͞ 13 C fractionation. It further predicts that, because a more product-like transition state is more ordered (decreased entropy), the effectiveness of this strategy will deteriorate with increasing temperature. enzyme mechanisms ͉ isotope fractionation ͉ transition states T he most abundant protein in nature is D-ribulose 1,5-bisphosphate (RuBP) carboxylase͞oxygenase (Rubisco, EC 4.1.1.39) (1). This immense N investment is required to counter the enzyme's pitifully sluggish catalytic performance. Furthermore, Rubisco's tendency to confuse the substrate of photosynthesis, CO 2 , with the product, O 2 , saddles all aerobic photosynthetic organisms with energy-wasting photorespiration (2). Thus, this single enzyme's efficiency, or lack thereof, dictates the (in)efficiency with which plants use their basic resources of light, water, and N, and currently intense biotechnological effort aims to improve its catalytic properties and to engineer such improvements into crop plants (3, 4). Rubisco's difficulties stem from the inevitable O 2 sensitivity of the 2,3-enediol form of RuBP, to which CO 2 is added during the carboxylase reaction (5), which causes carboxylase͞oxygenase bifunctionality (Fig. 1). This difficulty is exacerbated by the need to discriminate between featureless molecules, CO 2 and O 2 , that can be bound in Michaelis-Menten complexes only weakly, if at all (2, 6). R...

Discrimination against 13C during photosynthesis is a well-characterised phenomenon. It leaves behind distinct signatures in organic matter of plants and in the atmosphere. The former is depleted in 13C, the latter is enriched during periods of preponderant photosynthetic activity of terrestrial ecosystems. The intra-annual cycle and latitudinal gradient in atmospheric 13C resulting from photosynthetic and respiratory activities of terrestrial plants have been exploited for the reconstruction of sources and sinks through deconvolution by inverse modelling. Here, we compile evidence for widespread post-photosynthetic fractionation that further modifies the isotopic signatures of individual plant organs and consequently leads to consistent differences in delta13C between plant organs. Leaves were on average 0.96 per thousand and 1.91 per thousand more depleted than roots and woody stems, respectively. This phenomenon is relevant if the isotopic signature of CO2-exchange fluxes at the ecosystem level is used for the reconstruction of individual sources and sinks. It may also modify the parameterization of inverse modelling approaches if it leads to different isotopic signatures of organic matter with different residence times within the ecosystems and to a respiratory contribution to the average difference between the isotopic composition of plant organic matter and the atmosphere. We discuss the main hypotheses that can explain the observed inter-organ differences in delta13C.

The carbon isotope composition (␦ 13 C) of CO 2 produced in darkness by intact French bean (Phaseolus vulgaris) leaves was investigated for different leaf temperatures and during dark periods of increasing length. The ␦ 13 C of CO 2 linearly decreased when temperature increased, from Ϫ19‰ at 10°C to Ϫ24‰ at 35°C. It also progressively decreased from Ϫ21‰ to Ϫ30‰ when leaves were maintained in continuous darkness for several days. Under normal conditions (temperature not exceeding 30°C and normal dark period), the evolved CO 2 was enriched in 13 C compared with carbohydrates, the most 13 C-enriched metabolites. However, at the end of a long dark period (carbohydrate starvation), CO 2 was depleted in 13 C even when compared with the composition of total organic matter. In the two types of experiment, the variations of ␦ 13 C were linearly related to those of the respiratory quotient. This strongly suggests that the variation of ␦ 13 C is the direct consequence of a substrate switch that may occur to feed respiration; carbohydrate oxidation producing 13 C-enriched CO 2 and ␤-oxidation of fatty acids producing 13 C-depleted CO 2 when compared with total organic matter (Ϫ27.5‰). These results are consistent with the assumption that the ␦ 13 C of dark respired CO 2 is determined by the relative contributions of the two major decarboxylation processes that occur in darkness: pyruvate dehydrogenase activity and the Krebs cycle.Photosynthetic CO 2 assimilation of C 3 plants discriminates against 13 CO 2 so that organic matter is, on average, 20‰ depleted in 13 C compared with atmospheric carbon dioxide (for recent review, see Brugnoli and Farquhar, 2000). Respiratory carbon fluxes in light (i.e. photorespiration and "day" respiration) are often assumed to be negligible or weakly fractionating processes. However, the carbon isotope signature of organic matter may be modified by nighttime respiration depending on the ␦ 13 C of the evolved CO 2 because respiratory carbon lost by many plants has been shown to be within 30% to 60% of the carbon fixed through photosynthesis (Evans, 1993; Amthor, 2000).In vitro studies using protoplasts have shown that respired CO 2 isotope composition is identical to that of the Suc supplied to the culture medium, indicating that no fractionation occurs during respiration in the dark (Lin and Ehleringer, 1997). A similar result was also obtained in long-term experiments with animals, where the isotope composition of CO 2 expired by mice (Mus musculus) reflected that of the diet (Perkins and Speakman, 2001). In contrast, it has been shown previously that CO 2 produced by respiration in the dark is 6‰ 13 C enriched when compared with Suc in intact French bean (Phaseolus vulgaris) leaves (Duranceau et al., 1999). Similar results were also obtained in Nicotiana sylvestris and sunflower (Helianthus annuus), although CO 2 was less 13 C enriched with ␦ 13 C values of 4‰ and 3‰, respectively (Ghashghaie et al., 2001). Moreover, it has been demonstrated that the ␦ 13 C value of CO 2 evolved in the dar...

Non-photosynthetic, or heterotrophic, tissues in C3 plants tend to be enriched in 13C compared with the leaves that supply them with photosynthate. This isotopic pattern has been observed for woody stems, roots, seeds and fruits, emerging leaves, and parasitic plants incapable of net CO2 fixation. Unlike in C3 plants, roots of herbaceous C4 plants are generally not 13C-enriched compared with leaves. We review six hypotheses aimed at explaining this isotopic pattern in C3 plants: (1) variation in biochemical composition of heterotrophic tissues compared with leaves; (2) seasonal separation of growth of leaves and heterotrophic tissues, with corresponding variation in photosynthetic discrimination against 13C; (3) differential use of day v. night sucrose between leaves and sink tissues, with day sucrose being relatively 13C-depleted and night sucrose 13C-enriched; (4) isotopic fractionation during dark respiration; (5) carbon fixation by PEP carboxylase; and (6) developmental variation in photosynthetic discrimination against 13C during leaf expansion. Although hypotheses (1) and (2) may contribute to the general pattern, they cannot explain all observations. Some evidence exists in support of hypotheses (3) through to (6), although for hypothesis (6) it is largely circumstantial. Hypothesis (3) provides a promising avenue for future research. Direct tests of these hypotheses should be carried out to provide insight into the mechanisms causing within-plant variation in carbon isotope composition.