Carbon: terrestrial C4 plants

The carbon isotope composition of terrestrial C₄ plants depends on the primary carboxylation of phosphoenolpyruvate (PEP) and on the diffusion of CO₂ to the carboxylation sites, but is also influenced by the final carboxylation of ribulose-1,5-bisphosphate (RuBP). Several models have been used for reproducing this complex situation. In the present review, a particular model is applied as a means to interpret the effects of environmental and genetically determined factors on carbon isotope discrimination during C₄ photosynthesis. As a new feature, the model considers four types of limitation of the overall CO₂ assimilation rate. Both carboxylation reactions are assumed to be limited by either maximum enzyme activity or maximum substrate regeneration rate. The model is applied to experimental data on the effects of CO₂, irradiance and water stress on short-term discrimination by leaves of several C₄ species measured simultaneously with CO₂ gas exchange characteristics. In particular, different patterns of the influence of low irradiances on carbon isotope discrimination are interpreted as due to variations in that irradiance at which a transition from limitation by PEP regeneration rate and RuBP carboxylase activity to limitation by the regeneration rates of both substrates occurs. After discussing literature data on the effects of environmental conditions on carbon isotope discrimination by C₄ plants seasonal and developmental changes in carbon isotope composition, studies on the systematic and geographic distribution of C₄ plants, evolutionary and genetical aspects, and some ecological implications are reviewed.     

Carbon isotope composition of C4 grasses is influenced by light and water supply

The carbon isotope composition of C⁴ grasses has the potential to be used as an indicator of changes in the isotopic composition and concentration of atmospheric CO2, especially for climate reconstruction. The usefulness of C₄ grasses for this purpose hinges on the assumption that their photosynthetic discrimination against ¹³C remains constant in a wide range of environmental conditions. We tested this assumption by examining the effects of light and water stress on the carbon isotope composition of C₄ grasses using different biochemical subtypes (NADP-ME, NAD-ME, PCK) in glasshouse experiments. We grew 14 different C₄ grass species in four treatments: sun-watered, sun-drought, shade-watered and shade-drought. Carbon isotope discrimination (Δ) rarely remained constant. In general, Δ values were lowest in sun-watered grasses, greater for sun-drought plants and even higher for plants of the shade-watered treatment. The highest Δ values were generally found in the most stressed grasses, the shade-drought plants. Grasses of the NADP-ME subtype were the least influenced by a change in environmental variables, followed by PCK and NAD-ME subtypes. Water availability affected the carbon isotope discrimination less than light limitation in PCK and NAD-ME subtypes, but similarly in NADP-ME subtypes. In another experiment, we studied the effect of increasing light levels (150 to 1500 μmol photons m^?2 s^?1) on the Δ values of 18 well-watered C₄ grass species. Carbon isotope discrimination remained constant until photon flux density (PFD) was less than 700 μmol photons m^?2 s^?1. Below this light level, Δ values increased with decreasing irradiance for all biochemical subtypes. The change in A was less pronounced in NADP-ME and PCK than in NAD-ME grasses. Grasses grown in the field and in the glasshouse showed a similar pattern. Thus, caution should be exercised when using C4 plants under varying environmental conditions to monitor the concentration or carbon isotopic composition of atmospheric CO2 in field/glasshouse studies or climate reconstruction.     

A comparative study on the regulation of C3 and C4 carboxylation processes in the constitutive crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana and the C3-CAM intermediate Clusia minor

A comparison of carbon metabolism in the constitutive crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perr. and the C₃-CAM intermediate Clusia minor L. was undertaken under controlled environmental conditions where plants experience gradual changes in light intensity, temperature and humidity at the start and end of the photoperiod. The magnitude of CAM activity was manipulated by maintaining plants in ambient air or by enclosing leaves overnight in an atmosphere of N₂ to suppress C₄ carboxylation. Measurements of diel changes in carbonisotope discrimination and organic acid content were used to quantify the activities of C₃ and C₄ carboxylases in vivo and to indicate the extent to which the activities of phosphoenolpyruvate carboxylase (PEPCase), ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and decarboxylation processes overlap at the start and end of the photoperiod. These measurements in vivo were compared with measurements in vitro of changes in the diel sensitivity of PEPCase to malate inhibition. The results demonstrate fundamental differences in the down-regulation of PEPCase during the day in the two species. While PEPCase is inactivated within the first 30 min of the photoperiod in K. daigremontiana, the enzyme is active for 4 h at the start and 3 h at the end of the photoperiod in C. minor. Enclosing leaves in N₂ overnight resulted in a two-to threefold increase in PEPCase-mediated CO₂ uptake during Phase II of CAM in both species. However, futile cycling of CO₂ between malate synthesis and decarboxylation does not occur during Phase II in either species. In terms of overall carbon balance, C₄ carboxylation accounted for ≈ 20% of net daytime assimilation in both species under control conditions, increasing to 30–34% after a night in N₂. Although N₂-treated leaves of K. daigremontiana took up 25% more CO₂ than control leaves during the day this was insufficient to compensate for the loss of CO₂ taken up by CAM the previous night. In contrast, in N₂-treated leaves of C. minor, the twofold increase in daytime PEPCase activity and the increase in net CO₂ uptake by Rubisco during Phase III compensated for the inhibition of C₄ carboxylation at night in terms of diel carbon balance.     

Carbon isotope fractionation in plants

Plants with the C₃, C₄, and crassulacean acid metabolism (CAM) photosynthetic pathways show characteristically different discriminations against ¹³C during photosynthesis. For each photosynthetic type, no more than slight variations are observed within or among species. CAM plants show large variations in isotope fractionation with temperature, but other plants do not. Different plant organs, subcellular fractions and metabolises can show widely varying isotopic compositions. The isotopic composition of respired carbon is often different from that of plant carbon, but it is not currently possible to describe this effect in detail. The principal components which will affect the overall isotope discrimination during photosynthesis are diffusion of CO₂, interconversion of CO₂ and HCO^?₃, incorporation of CO₂ by phosphoenolpyruvate carboxylase or ribulose bisphosphate carboxylase, and respiration. Theisotope fractionations associated with these processes are summarized. Mathematical models are presented which permit prediction of the overall isotope discrimination in terms of these components. These models also permit a correlation of isotope fractionations with internal CO₂ concentrations. Analysis of existing data in terms of these models reveals that CO₂ incorporation in C₃ plants is limited principally by ribulose bisphosphate carboxylase, but CO₂ diffusion also contributes. In C₄ plants, carbon fixation is principally limited by the rate of CO₂ diffusion into the leaf. There is probably a small fractionation in C₄ plants due to ribulose bisphosphate carboxylase.     

Isotope ratios of cellulose from plants having different photosynthetic pathways

Hydrogen and carbon isotope ratios of cellulose nitrate and oxygen isotope ratios of cellulose from C₃, C₄, and Crassulacean acid metabolism (CAM) plants were determined for plants growing within a small area in Val Verde County, Texas. Plants having CAM had distinctly higher deuterium/hydrogen (D/H) ratios than plants having C₃ and C₄ metabolism. When hydrogen isotope ratios are plotted against carbon isotope ratios, each photosynthetic mode separates into a distinct cluster of points. C₄ plants had many D/H ratios similar to those of C₃ plants, so that hydrogen isotope ratios cannot be used to distinguish between these two photosynthetic modes. Portulaca mundula, which may have a modified photosynthetic mode between C₄ and CAM, had a hydrogen isotope ratio between those of the C₄ and CAM plants. When oxygen isotope ratios are plotted against carbon isotope ratios, no distinct clustering of the C₄ and CAM plants occurs. Thus, oxygen isotope ratios are not useful in distinguishing between these metabolic modes. A plot of hydrogen isotope ratios versus oxygen isotope ratios for this sample set shows considerable overlap between oxygen isotope ratios of the different photosynthetic modes without a concomitant overlap in the hydrogen isotope ratios of CAM and the other two photosynthetic modes. This observation is consistent with the hypothesis that higher D/H ratios in CAM plants relative to C₃ and C₄ plants are due to isotopic fractionations occurring during biochemical reactions.     

Species-specific differences in temporal and spatial variation in δ13C of plant carbon pools and dark-respired CO2 under changing environmental conditions

Stable carbon isotope signatures are often used as tracers for environmentally driven changes in photosynthetic δ¹³C discrimination. However, carbon isotope signatures downstream from carboxylation by Rubisco are altered within metabolic pathways, transport and respiratory processes, leading to differences in δ¹³C between carbon pools along the plant axis and in respired CO₂. Little is known about the within-plant variation in δ¹³C under different environmental conditions or between species. We analyzed spatial, diurnal, and environmental variations in δ¹³C of water soluble organic matter (δ¹³C_WSOM) of leaves, phloem and roots, as well as dark-respired δ¹³CO₂ (δ¹³C_res) in leaves and roots. We selected distinct light environments (forest understory and an open area), seasons (Mediterranean spring and summer drought) and three functionally distinct understory species (two native shrubs—Halimium halimifolium and Rosmarinus officinalis—and a woody invader—Acacia longifolia). Spatial patterns in δ¹³C_WSOM along the plant vertical axis and between respired δ¹³CO₂ and its putative substrate were clearly species specific and the most δ¹³C-enriched and depleted values were found in δ¹³C of leaf dark-respired CO₂ and phloem sugars, ~?15 and ~?33 ‰, respectively. Comparisons between study sites and seasons revealed that spatial and diurnal patterns were influenced by environmental conditions. Within a species, phloem δ¹³C_WSOM and δ¹³C_res varied by up to 4 ‰ between seasons and sites. Thus, careful characterization of the magnitude and environmental dependence of apparent post-carboxylation fractionation is needed when using δ¹³C signatures to trace changes in photosynthetic discrimination.     

Use of decreasing foliar carbon isotope discrimination during water limitation as a carbon tracer to study whole plant carbon allocation

Foliar carbon isotope discrimination (Δ) of C₃ plants decreases in water‐deficit situations as discrimination by the photosynthetic primary carboxylation reaction decreases. This diminished Δ in leaves under water deficit can be used as a tracer to study whole plant carbon allocation patterns. Carbon isotope composition (δ¹³C value) of leaf hot water extracts or leaf tissue sap represents a short‐term integral of leaf carbon isotope discrimination and thus represents the δ¹³C value of source carbon that may be distributed within a plant in water‐deficit situations. By plotting the δ¹³C values of source carbon against the δ¹³C values of sink tissues, such as roots or stems, it is possible to assess carbon allocation to and incorporation into sink organs in relation to already present biomass. This natural abundance labelling method has been tested in three independent experiments, a one‐year field study with the fruit tree species Ziziphus mauritiana and peach (Prunus persica), a medium‐term drought stress experiment with Ziziphus rotundifolia trees in the glasshouse, and a short‐term drought stress experiment with soybean (Glycine max). The data show that the natural abundance labelling method can be applied to qualitatively assess carbon allocation in drought‐stressed plants. Although it is not possible to estimate exact fluxes of assimilated carbon during water deficit the method represents an easy to use tool to study integrated plant adaptations to drought stress. In addition, it is a less laborious method that can be applied in field studies as well as in controlled experiments, with plants from any developmental stage.     

The effects of salinity,crassulacean acid metabolism and plant age on the carbon isotope composition of <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> L., a halophytic C<Subscript>3</Subscript>-CAM species

The carbon isotope composition of the halophyte Mesembryanthemum crystallinum L. (Aizoaceae) changes when plants are exposed to environmental stress and when they shift from C₃ to crassulacean acid metabolism (CAM). We examined the coupling between carbon isotope composition and photosynthetic pathway by subjecting plants of different ages to salinity and humidity treatments. Whole shoot ¹³C values became less negative in plants that were exposed to 400 mM NaCl in the hydroponic solution. The isotopic change had two components: a direct NaCl effect that was greatest in plants still operating in the C₃ mode and decreased proportionally with increasing levels of dark fixation, and a second component related to the degree of CAM expression. Ignoring the presumably diffusion-related NaCl effect on carbon isotope ratios results in an overestimation of nocturnal CO₂ gain in comparison to an isotope versus nocturnal CO₂ gain calibration established previously for C₃ and CAM species grown under well-watered conditions. It is widely taken for granted that the shift to CAM in M. crystallinum is partially under developmental control and that CAM is inevitably expressed in mature plants. Plants, cultivated under non-saline conditions and high relative humidity (RH) for up to 63 days, maintained diel CO₂ gas-exchange patterns and ¹³C values typical of C₃ plants. However, a weak CAM gas-exchange pattern and an increase in ¹³C value were observed in non-salt-treated plants grown at reduced RH. These observations are consistent with environmental control rather than developmental control of the induction of CAM in mature M. crystallinum under non-saline conditions.     

Relative humidity- and ABA-induced variation in carbon and oxygen isotope ratios of cotton leaves

Cotton (Gossypium hirsutum L. cv. CS50) plants were grown at two levels of relative humidity (RH) and sprayed daily with abscisic acid (ABA) at four concentrations. Plants grown at lower humidity had higher transpiration rates, lower leaf temperatures and lower stomatal conductance. Plant biomass was also reduced at low humidity. Within each humidity environment, increasing ABA concentration generally reduced stomatal conductance, evaporation rates, superficial leaf density and plant biomass, and increased leaf temperature and specific leaf area. As expected, decreased stomatal conductance resulted in decreased carbon isotope discrimination in leaf material ( Δ ¹³C_l). Plants grown at low humidity were more enriched in ¹⁸O than those grown at high RH, as theory predicts. Within each humidity environment, increasing ABA concentration increased oxygen isotope enrichment of leaf cellulose ( Δ ¹⁸O_c) and whole‐leaf tissue ( Δ ¹⁸O_l). Values of Δ ¹³C_l and Δ ¹⁸O_l predicted by theoretical models were close to those observed, accounting for 79% of the measured variation in Δ ¹³C_l and 95% of the measured variation in Δ ¹⁸O_l. Supporting theory, Δ ¹³C_l and Δ ¹⁸O_l in whole‐leaf tissue were negatively related.     

Seasonal influences on carbohydrate metabolism in the CAM bromeliad Aechmea ��Maya��: consequences for carbohydrate partitioning and growth

Background and Aims

Photosynthetic plasticity in response to a range of environmental factors that include [CO₂], water availability, light intensity and temperature, is ubiquitous among plants with crassulacean acid metabolism (CAM). The present study examined how seasonal changes in light availability, as experienced by greenhouse CAM crops in northern latitude regions, influence diel carboxylation patterns and impact on carbon gain and seasonal accumulation of biomass.

Methods

In the CAM bromeliad Aechmea ‘Maya’ integrated measurements of leaf gas exchange, diel metabolite dynamics (e.g. malate, soluble sugars and starch) and biomass accumulation were made four times a year, i.e. in winter, spring, summer and autumn.

Key Results

During the brighter seasons (spring and summer) daytime Phases II and IV were dominated by C₄ carboxylation, whilst the higher diurnal uptake in the autumn and winter was characterized by equal contributions of both Rubisco and PEPC. As a consequence, net CO₂ uptake showed a significant depression at the end of the day in the darker months when supplementary illumination was turned off. Remarkable seasonal consistency was found in the amount of storage reserves available for nocturnal carboxylation, a consequence of predominantly daytime export of carbohydrate in spring and summer whilst nocturnal export was the major sink for carbohydrate in autumn and winter.

Conclusions

Throughout the different seasons Aechmea ‘Maya’ showed considerable plasticity in the timing and magnitude of C₃ and C₄ carboxylation processes over the diel cycle. Under low PPFD (i.e. winter and autumn) it appears that there was a constraint on the amount of carbohydrate exported during the day in order to maintain a consistent pool of transient carbohydrate reserves. This gave remarkable seasonal consistency in the amount of storage reserves available at night, thereby optimizing biomass gain throughout the year. The data have important practical consequences for horticultural productivity of CAM plants and suggest a scenario for reconciling carbohydrate partitioning between competing sinks of nocturnal acidification and export for growth.Key words: Aechmea ‘Maya’, seasonal, CAM, bromeliad, carbon budgets, gas exchange, metabolite dynamics, PEPC, photoperiod extension, PPFD, photosynthetic plasticity, Rubisco     

Carbon, hydrogen, and oxygen isotope ratios of cellulose from plants having intermediary photosynthetic modes

Carbon and hydrogen isotope ratios of cellulose nitrate and oxygen isotope ratios of cellulose from species of greenhouse plants having different photosynthetic modes were determined. When hydrogen isotope ratios are plotted against carbon isotope ratios, four clusters of points are discernible, each representing different photosynthetic modes: C₃ plants, C₄ plants, CAM plants, and C₃ plants that can shift to CAM or show the phenomenon referred to as CAM-cycling. The combination of oxygen and carbon isotope ratios does not distinguish among the different photosynthetic modes. Analysis of the carbon and hydrogen isotope ratios of cellulose nitrate should prove useful for screening different photosynthetic modes in field specimens that grew near one another. This method will be particularly useful for detection of plants which show CAM-cycling.     

Combining meteorology, eddy fluxes, isotope measurements, and modeling to understand environmental controls of carbon isotope discrimination at the canopy scale

Estimates of terrestrial carbon isotope discrimination are useful to quantify the terrestrial carbon sink. Carbon isotope discrimination by terrestrial ecosystems may vary on seasonal and interannual time frames, because it is affected by processes (e.g. photosynthesis, stomatal conductance, and respiration) that respond to variable environmental conditions (e.g. air humidity, temperature, light). In this study, we report simulations of the temporal variability of canopy‐scale C₃ photosynthetic carbon isotope discrimination obtained with an ecophysiologically based model (ISOLSM) designed for inclusion in global models. ISOLSM was driven by half‐hourly meteorology, and parameterized with eddy covariance measurements of carbon and energy fluxes and foliar carbon isotope ratios from a pine forest in Metolius (OR). Comparing simulated carbon and energy fluxes with observations provided a range of parameter values that optimized the simulated fluxes. We found that the sensitivity of photosynthetic carbon isotope discrimination to the slope of the stomatal conductance equation (m, Ball–Berry constant) provided an additional constraint to the model, reducing the wide parameter space obtained from the fluxes alone. We selected values of m that resulted in similar simulated long‐term discrimination as foliar isotope ratios measured at the site. The model was tested with ¹³C measurements of ecosystem (δ_R) and foliar (δ_f) respiration. The daily variability of simulated ¹³C values of assimilated carbon (δ_A) was similar to that of observed δ_f, and higher than that of observed and simulated δ_R. We also found similar relationships between environmental factors (i.e. vapor pressure deficit) and simulated δ_R as measured in ecosystem surveys of δ_R. Therefore, ISOLSM reasonably simulated the short‐term variability of δ_A controlled by atmospheric conditions at the canopy scale, which can be useful to estimate the variability of terrestrial isotope discrimination. Our study also shows that including the capacity to simulate carbon isotope discrimination, together with simple ecosystem isotope measurements, can provide a useful constraint to land surface and carbon balance models.     

HERITABILITY OF CARBON ISOTOPE DISCRIMINATION IN GUTIERREZIA MICROCEPHALA (ASTERACEAE)

The carbon isotope composition (¹³C/¹²C) of C₃ plant tissues provides a long-term, integrated measure of photosynthetic metabolism. Quantitative genetic methods were used to study the inheritance of carbon isotope composition and several morphological characters in Gutierrezia microcephala, a short-lived desert perennial. Open-pollinated seed was collected from a population located near Lee's Ferry, Arizona, in an area that was disturbed approximately 20 years ago. Seeds were germinated and seedlings grown in a common greenhouse environment. Carbon isotope discrimination (Δ) and all morphological characters varied significantly among maternal families. Heritability of Δ was estimated as 81% in the greenhouse and as 92% using the regression of family mean Δ on parent Δ from field samples. Using both field and greenhouse data, we estimated a lower bound for heritability in nature of 54%. Offspring size and biomass both showed significant, positive correlations with Δ. The corresponding genetic correlations were similar in magnitude and direction, but did not differ significantly from zero. These results imply the existence of heritable differences in physiology associated with carbon assimilation and water loss within populations, and the potential for microevolutionary change through natural selection.     

Carbon isotope discrimination in C3-C4 intermediates

Carbon isotope discrimination in C₃–C₄ intermediates is determined by fractionations during diffusion and the biochemical fractionations occurring during CO₂ fixation. These biochemical fractionations in turn depend on the fractionation by Rubisco in the mesophyll, the amount of CO₂ fixation. These biochemical fractionations in turn depend on the fractionation by Rubisco in the mesophyll, the amount of CO₂ fixation occurring in the bundle sheath, the extent of bundle-sheath leakiness and the contribution which C₄-cycle activity makes to the CO₂ pool there. In most instances, carbon isotope discrimination in C₃–C₄ intermediates is C₃-like because only a small fraction of the total carbon fixed is fixed in the bundle sheath. In particular, this must be the case for Flaveria intermediates which initially fix substantial amounts of CO₂ into C₄-acids. In C₃–C₄ intermediates that refix photorespiratory CO₂ alone, it is possible for carbon isotope discrimination to be greater than in C₃-species, particularly at low CO₂ pressures or at high leaf temperatures. Short-term measurements of carbon isotope discrimination and gas exchange of leaves can be used to study the photosynthetic pathways of C₃-C₄ intermediates and their hybrids as has recently been done for C₃ and C₄ species.     

Diel shifts in carboxylation pathway and metabolite dynamics in the CAM bromeliad Aechmea 'Maya' in response to elevated CO2

Background and Aims

The deployment of temporally separated carboxylation pathways for net CO₂ uptake in CAM plants provides plasticity and thus uncertainty on how species with this photosynthetic pathway will respond to life in a higher-CO₂ world. The present study examined how long-term exposure to elevated CO₂ influences the relative contributions that C₃ and C₄ carboxylation make to net carbon gain and to establish how this impacts on the availability of carbohydrates for export and growth and on water use efficiency over the day/night cycle.

Methods

Integrated measurements of leaf gas exchange and diel metabolite dynamics (e.g. malate, soluble sugars, starch) were made in leaves of the CAM bromeliad Aechmea ‘Maya’ after exposure to 700 µmol mol⁻¹ CO₂ for 5 months.

Key Results

There was a 60 % increase in 24-h carbon gain under elevated CO₂ due to a stimulation of daytime C₃ and C₄ carboxylation in phases II and IV where water use efficiency was comparable with that measured at night. The extra CO₂ taken up under elevated CO₂ was largely accumulated as hexose sugars during phase IV and net daytime export of carbohydrate was abolished. Under elevated CO₂ there was no stimulation of dark carboxylation and nocturnal export and respiration appeared to be the stronger sinks for carbohydrate.

Conclusions

Despite the increased size of the soluble sugar storage pool under elevated CO₂, there was no change in the net allocation of carbohydrates between provision of substrates for CAM and export/respiration in A. ‘Maya’. The data imply the existence of discrete pools of carbohydrate that provide substrate for CAM or sugars for export/respiration. The 2-fold increase in water-use efficiency could be a major physiological advantage to growth under elevated CO₂ in this CAM bromeliad.Key words: Aechmea ‘Maya’, carbon budgets, elevated CO₂, gas exchange, metabolite dynamics, PEPC, photosynthetic plasticity, Rubisco     

Short-term changes in carbon-isotope discrimination in the C3-CAM intermediate Clusia minor L. growing in Trinidad

On-line instantaneous carbon isotope discrimination was measured in conjunction with net uptake of CO₂ in leaves of exposed and shaded plants of the C₃-CAM intermediate Clusia minor growing under natural conditions in Trinidad. At the end of the rainy season (late January-early February, 1992) C₃ photosynthesis predominated although exposed leaves recaptured a small proportion of respiratory CO₂ at night for the synthesis of malic acid. Citric acid was the major organic acid accumulated by exposed leaves at this time with a citric: malic acid ratio of 11:1. Values of instantaneous discrimination () in exposed leaves during the wet season rose from 17.1 shortly after dawn to 22.7 around mid-day just before stomata closed, suggesting that most CO₂ was fixed by Rubisco at this time. During the late afternoon, instantaneous declined from 22.2 to 17, probably reflecting the limited contribution from PEPc activity and an increase in diffusional resistance to CO₂ in exposed leaves. Shaded leaves showed no CAM activity and CO₂ uptake proceeded throughout the day in the wet season. The decrease in instantaneous from 27 in the morning to 19.2 in the late afternoon was therefore entirely due to diffusional limitation. Leaves sampled in the dry season (mid-March, 1992) had by now induced full CAM activity with both malic and citric acids accumulated overnight and stomata closed for 4–5 h over the middle of the day. Values of instantaneous measured over the first 3 h after dawn (6.4–9.1) indicated that C₄ carboxylation dominated CO₂ uptake for most of the morning when rates of photosynthesis were maximal, implying that under natural conditions, the down regulation of PEPc in phase II occurs much more slowly than laboratory-based studies have suggested. The contribution from C₃ carboxylation to CO₂ uptake during phase II was most marked in leaves which accumulated lower quantities of organic acids overnight. In exposed leaves, measurements of instantaneous during the late afternoon illustrated the transition from C₃ to C₄ carboxylation with stomata remaining open during the transition from dusk into the dark period. Uptake of CO₂ by shaded leaves during the late afternoon however appeared to be predominantly limited by decreased stomatal conductance. The short-term measurements of instantaneous were subsequently integrated over 24 h in order to predict the leaf carbon isotope ratios (_p) and to compare this with the _p measured for leaf organic material. Whilst there was close agreement between predicted and measured _p for plants sampled in the wet season, during the dry season the predicted carbon isotope ratios were 5–9 higher than the measured isotope ratios. During the annual cycle of leaf growth most carbon was fixed via the C₃ pathway although CAM clearly plays an important role in maintaining photochemical integrity in the dry season.     

Photorespiration Contributes to Stomatal Regulation and Carbon Isotope Fractionation: A Study with Barley,Potato and Arabidopsis Plants Deficient in Glycine Decarboxylase

The rates of respiration in light and darkness, C _i/C _a and carbon isotope fractionation were investigated in glycine decarboxylase-deficient plants of barley, potato and Arabidopsis thaliana grown in climate chambers with controlled light intensity, temperature, humidity, irradiation and different CO₂ concentrations (360, 700 and 1400 µl l^–1) and compared to the wild-type plants. All photorespiration-impaired plants exhibited higher C _i/C _a and corresponding lower apparent water-use efficiencies, which were more expressed under high irradiance and elevated temperature. The mutants were depleted in ¹³C as compared to the wild-type plants, with a difference of up to 6 following growth in 360 µl l^–1 CO₂. We determined the carbon isotope content at different CO₂ concentrations to calculate the contribution of both C _i/C _a and photorespiration for ¹³C/¹²C fractionation. The direct effect of photorespiration was in the range of 0.7–1.0, from which we calculated the value of fractionation at the site of glycine decarboxylation as being 10–13, which is in agreement with the previously reported carbon isotope discrimination exerted by the glycine decarboxylase. Respiratory rates, particularly in the light, were increased in the glycine decarboxylase mutants. The necessity of the maintenance of a high CO₂ concentration near the site of carboxylation in chloroplasts in plants deficient in photorespiratory enzymes, requires an increased opening of the stomata with a corresponding decrease in water-use efficiency. It is concluded that photorespiration participates in the regulation of C _i/C _a and contributes to carbon isotope fractionation, both via effects on stomata and via discrimination of ¹³C in the glycine decarboxylase reaction.This revised version was published online in October 2005 with corrections to the Cover Date.     

Stable hydrogen isotope ratios of saponifiable lipids and cellulose nitrate from cam,c3 and c4 plants

Hydrogen and carbon isotope ratios of saponifiable lipids and cellulose nitrate from CAM, C₃, and C₄ plants that grew near one another were determined. The deuterium/protium (D/H) ratios of cellulose nitrate from CAM plants were much higher than those of cellulose nitrate from C₃ and C₄ plants, as has been observed previously. In contrast, the D/H ratios of saponifiable lipids from CAM plants did not differ from those of the same fraction from C₃ and C₄ plants. These observations indicate that deuterium enrichment in cellulose of CAM plants is not caused by any metabolic or physiological process which would lead to deuterium enrichment in all biochemical fractions.     

Effects of RuBisCO and CO2 concentration on cyanobacterial growth and carbon isotope fractionation

Carbon isotope biosignatures preserved in the Precambrian geologic record are primarily interpreted to reflect ancient cyanobacterial carbon fixation catalyzed by Form I RuBisCO enzymes. The average range of isotopic biosignatures generally follows that produced by extant cyanobacteria. However, this observation is difficult to reconcile with several environmental (e.g., temperature, pH, and CO₂ concentrations), molecular, and physiological factors that likely would have differed during the Precambrian and can produce fractionation variability in contemporary organisms that meets or exceeds that observed in the geologic record. To test a specific range of genetic and environmental factors that may impact ancient carbon isotope biosignatures, we engineered a mutant strain of the model cyanobacterium Synechococcus elongatus PCC 7942 that overexpresses RuBisCO across varying atmospheric CO₂ concentrations. We hypothesized that changes in RuBisCO expression would impact the net rates of intracellular CO₂ fixation versus CO₂ supply, and thus whole-cell carbon isotope discrimination. In particular, we investigated the impacts of RuBisCO overexpression under changing CO₂ concentrations on both carbon isotope biosignatures and cyanobacterial physiology, including cell growth and oxygen evolution rates. We found that an increased pool of active RuBisCO does not significantly affect the ¹³C/¹²C isotopic discrimination (ε_p) at all tested CO₂ concentrations, yielding ε_p of ≈ 23‰ for both wild-type and mutant strains at elevated CO₂. We therefore suggest that expected variation in cyanobacterial RuBisCO expression patterns should not confound carbon isotope biosignature interpretation. A deeper understanding of environmental, evolutionary, and intracellular factors that impact cyanobacterial physiology and isotope discrimination is crucial for reconciling microbially driven carbon biosignatures with those preserved in the geologic record.     

Intraspecific variation in photosynthetic responses of trebouxioid lichens with reference to the activity of a carbon-concentrating mechanism

The photosynthetic responses of a range of trebouxioid lichens were investigated to determine whether variations in net assimilation rates shown by populations of the same species collected from different habitats could be correlated with adjustments in carbon-concentrating mechanism (CCM) activity. The activity of a CCM was inferred from the high affinity for CO₂ [i.e. low CO₂ compensation point (Γ); low external CO₂ concentration at which half-maximal assimilation rates are reached (K _{0.5 CO2})], the release of a pool of accumulated dissolved inorganic carbon (C_i) during light/dark transient measurements of CO₂ exchange and values for carbon isotope discrimination intermediate between those characteristic of C₃ and C₄ terrestrial plants. Higher net and gross assimilation rates were expressed by lichens collected from shaded woodland habitats. The higher rates were not accounted for by variations in chlorophyll content. Lichens with high assimilation rates also showed an increased affinity for CO₂ as demonstrated by low CO₂ compensation points and K _0.5 values and the magnitude of the C_i pool accumulated upon illumination and released after darkening of the thalli. However, there was no correlation between assimilation rates and organic matter or instantaneous carbon isotope discrimination measurements, with the latter remaining roughly consistent whatever the provenance or species of the lichen material. The data are discussed with reference to significant environmental factors which are likely to control photosynthesis in the habitats studied. Received: 5 April 1997 / Accepted: 9 September 1997