Comparative ecophysiology of C3 and C4 plants

Abstract. In this review we relate the physiological significance of C₄ photosynthesis to plant performance in nature. We begin with an examination of the physiological consequences of the C₄ pathway on photosynthesis, then discuss the ecophysiological performance of C₄ plants in contrasting environments. We then compare the performance of C₃ and C₄ plants when they occur together in similar habitats, and finally discuss the distribution of C₄ photosynthesis with respect to the physical environment, phylogeny, and life form.     

Compatible solutes and their effects on phosphoenolpyruvate carboxylase of C4-halophytes

Abstract Phosphoenolpyruvate carboxylase (PEPCase), extracted from two Poaceae (Cynodon dactylon and Sporobolus pungens) grown on saline soil, was affected physiologically by betaine and proline. Its affinity for phosphoenolpyruvate (PEP) was increased and full protection against NaCl inhibition was observed; enzymic activity was also stabilized when assayed at low PEP levels. Betaine has the same effects on PEPCase extracted from a Chenopodiaceae (Salsola soda), whereas proline behaves as a competitive inhibitor, i.e. it does not protect the enzyme against NaCl and it accelerates inactivation at low PEP levels. Betaine was only compatible with PEPCase extracted from Saisola kali, without any effect on activity, protection or stabilization, but proline was again inhibitory. The levels of free proline in the two salt-stressed Poaceae were high, whereas in the Chenopodiaceae the free proline was low, as in non-stressed plants. The above data indicate that osmoregulators could not only be compatible with cytoplasmic enzymes, but they could either promote or inhibit enzyme activity, depending on the source of enzyme. Coevolution of PEPCase with the osmolyte selected for, could also be inferred.     

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.     

Comparison of the specific activity of ribulose-1,5-bis-phosphate carboxylase-oxygenase from some C3 and C4 plants

The specific activity of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco, EC 4.1.1.39) was measured from the crude extracts of five C₃ plants consisting of wheat ( Triticum aestivum L. cv. Maris Mink), spinach ( Spinacia oleracea L.), pea ( Pisum sativum L. cv. Greenfeast), pumpkin ( Cucurbita pepo L. cv. Jättiläismeloni) and Ceratodon purpureus (Hedw.) Brid., and two C₄ plants, maize ( Zea mays L. ETA F₁) and sugar sorghum [ Sorghum saccharatum (L. emend, L.) Moench]. The amount of Rubisco in the crude extracts was estimated by polyacrylamide gel electro-phoresis with the Coomassie Brilliant Blue staining procedure. The amounts of the dye bound to the purified Rubisco of different higher plants were similar. The method gave a linear response for both purified enzyme and crude extracts, and the results agreed with those observed by immunochemical methods. The addition of positive effectors such as inorganic phosphate was necessary to obtain maximal activity in the crude extracts of all the studied plants except in that of maize. No significant differences in the specific carboxylase activity at 25°C were found between the C₃ and C₄ plants.     

Carbonic anhydrase in the plasma membranes from leaves of C3 and C4 plants

Plasma membranes were isolated from green leaves of maize ( Zea mays ), spinach ( Spinacia oleracea ), Setaria viridis and wheat ( Triticum aestivum cv. Omase) by aqueous two-phase partitioning. Carbonic anhydrase activity was detected in these membranes. The activity was inhibited by specific inhibitors for carbonic anhydrase, acetazolamide and ethoxyzolamide. The carbonic anhydrase activity was markedly enhanced by the addition of Triton X-100 to the plasma membranes. The highest activity was obtained in the presence of 0.015% detergent. The activity was scarcely affected when the plasma membrane vesicles were treated with proteinase K, but largely inactivated by the protease after treating the membranes with Triton X-100. These results indicate that carbonic anhydrase faces the cytoplasmic side of the membrane since plasma membranes purified by aqueous two-phase partitioning are tightly sealed vesicles of right side-out orientation (apoplastic side-out). With leaves of C₄ plants, 20 to 60% of the total carbonic anhydrase activity was found in the microsomal fraction. By contrast, only 1 to 3% of the activity was found in the microsomal fraction from leaves of C₃ plants. Western blot analysis showed that a polypeptide in the spinach plasma membrane cross-reacted with an antiserum raised against spinach chloroplast carbonic anhydrase, and that the molecular mass of the plasma membrane enzyme was higher than that of the chloroplast carbonic anhydrase (28 and 26 kDa, respectively). This indicates the presence of different molecular species of carbonic anhydrase in the chloroplast and the plasma membrane.     

Immunological studies on phosphoenolpyruvate carboxylase in Sedum species with crassulacean acid metabolism or C3 photosynthesis

Abstract From Sedum morganianum, which is a plant species known to have constitutive crassulacean acid metabolism (CAM), phosphoenolpyruvate (PEP) carboxylase (E.C.4.1.1.31) has been extracted and purified by (NH₄)₂SC₄ precipitation, ion exchange chromatography and gel electrophoresis. A specific antibody to this purified enzyme was obtained by immunization of a rabbit. This antibody was used to compare the antigen–antibody reaction of PEP-carboxylases prepared from other Sedum species including constitutive, facultative and non-CAM plants. The experiments revealed partial immunological indentity of PEP-carboxylases obtained from the different sources.     

Naturally low carbonic anhydrase activity in C4 and C3 plants limits discrimination against C18OO during photosynthesis

The ¹⁸O content of CO₂ is a powerful tracer of photosynthetic activity at the ecosystem and global scale. Due to oxygen exchange between CO₂ and ¹⁸O-enriched leaf water and retrodiffusion of most of this CO₂ back to the atmosphere, leaves effectively discriminate against ¹⁸O during photosynthesis. Discrimination against ¹⁸O ( Δ ¹⁸O) is expected to be lower in C₄ plants because of low c_i and hence low retrodiffusing CO₂ flux. C₄ plants also generally show lower levels of carbonic anhydrase (CA) activities than C₃ plants. Low CA may limit the extent of ¹⁸O exchange and further reduce Δ ¹⁸O. We investigated CO₂–H₂O isotopic equilibrium in plants with naturally low CA activity, including two C₄ (Zea mays, Sorghum bicolor) and one C₃ (Phragmites australis) species. The results confirmed experimentally the occurrence of low Δ ¹⁸O in C₄, as well as in some C₃, plants. Variations in CA activity and in the extent of CO₂–H₂O isotopic equilibrium ( θ _eq) estimated from on-line measurements of Δ ¹⁸O showed large range of 0–100% isotopic equilibrium ( θ _eq = 0–1). This was consistent with direct estimates based on assays of CA activity and measurements of CO₂ concentrations and residence times in the leaves. The results demonstrate the potential usefulness of Δ ¹⁸O as indicator of CA activity in vivo. Sensitivity tests indicated also that the impact of θ _eq < 1 (incomplete isotopic equilibrium) on ¹⁸O of atmospheric CO₂ can be similar for C₃ and C₄ plants and in both cases it increases with natural enrichment of ¹⁸O in leaf water.     

Models of carbon metabolism in C3-C4 intermediate plants as applied to the evolution of C4 photosynthesis

Abstract Models developed to explain the biphasic response of CO₂ compensation concentration to O₂ concentration and the C₃-like carbon isotope discrimination in C₃-C₄ intermediate species are used to characterize quantitatively the steps necessary in the evolution of C₄ photosynthesis. The evolutionary stages are indicated by model outputs, CO₂ compensation concentration and δ₁₃C value. The transition from intermediate plants to C₄ plants requires the complete formation of C₄ cycle capacity, expressed by the models as transition from C₄ cycle limitation by phosphoenolpyruvate (PEP) regeneration rate to limitation by PEP carboxylase activity. Other steps refer to CO₂ leakage from bundle sheath cells, to further augmentations of C₄ cycle components, to the repression of ribulose-1,5-bisphos-phate carboxylase in the mesophyll cells, and to a decrease in the CO₂ affinity of the enzyme. Possibilities of extending the suggested approach to other physiological characteristics, and the adaptive significance of the steps envisaged, are discussed.     

Mesophyll cell properties for some C3 and C4 species with high photosynthetic rates

Leaves of twelve C₃ species and six C₄ species were examined to understand better the relationship between mesophyll cell properties and the generally high photosynthetic rates of these plants. The CO₂ diffusion conductance expressed per unit mesophyll cell surface area (g_CO2^cell) cell was determined using measurements of the net rate of CO₂ uptake, water vapor conductance, and the ratio of mesophyll cell surface area to leaf surface area (A^mes/A). A^mes/A averaged 31 for the C₃ species and 16 for the C₄ species. For the C₃ species g_CO2^cell ranged from 0.12 to 0.32 mm s^-1, and for the C₄ species it ranged from 0.55 to 1.5 mm s^-1, exceeding a previously predicted maximum of 0.5 mm s^-1. Although the C₃ species Cammissonia claviformis did not have the highest g_CO2^cell, the combination of the highest A^mes and highest stomatal conductance resulted in this species having the greatest maximum rate of CO₂ uptake in low oxygen, 93 μmol m^-2 s^-1 (147 mg dm^-2 h^-1). The high g_CO2^cell of the C₄ species Amaranthus retroflexus (1.5 mm s^-1) was in part attributable to its thin cell wall (72 nm thick).     

On the significance of C3—C4 intermediate photosynthesis to the evolution of C4 photosynthesis

Abstract Evidence is drawn from previous studies to argue that C₃—C₄ intermediate plants are evolutionary intermediates, evolving from fully-expressed C₃ plants towards fully-expressed C₄ plants. On the basis of this conclusion, C₃—C₄ intermediates are examined to elucidate possible patterns that have been followed during the evolution of C₄ photosynthesis. An hypothesis is proposed that the initial step in C₄-evolution was the development of bundle-sheath metabolism that reduced apparent photorespiration by an efficient recycling of CO₂ using RuBP carboxylase. The CO₂-recycling mechanism appears to involve the differential compartmentation of glycine decarboxylase between mesophyll and bundle-sheath cells, such that most of the activity is in the bundlesheath cells. Subsequently, elevated phosphoenolpyruvate (PEP) carboxylase activities are proposed to have evolved as a means of enhancing the recycling of photorespired CO₂. As the activity of PEP carboxylase increased to higher values, other enzymes in the C₄-pathway are proposed to have increased in activity to facilitate the processing of the products of C₄-assimilation and provide PEP substrate to PEP carboxylase with greater efficiency. Initially, such a ‘C₄-cycle’ would not have been differentially compartmentalized between mesophyll and bundlesheath cells as is typical of fully-expressed C₄ plants. Such metabolism would have limited benefit in terms of concentrating CO₂ at RuBP carboxylase and, therefore, also be of little benefit for improving water- and nitrogen-use efficiencies. However, the development of such a limited C₄-cycle would have represented a preadaptation capable of evolving into the leaf biochemistry typical of fully-expressed C₄ plants. Thus, during the initial stages of C₄-evolution it is proposed that improvements in photorespiratory CO₂-loss and their influence on increasing the rate of net CO₂ assimilation per unit leaf area represented the evolutionary ‘driving-force’. Improved resourceuse efficiency resulting from an efficient CO₂-concentrating mechanism is proposed as the driving force during the later stages.     

Zeaxanthin and non‐photochemical quenching in sun and shade leaves of C3 and C4 plants

The relationships between non‐radiative energy dissipation and the carotenoid content, especially the xanthophyll cycle components, were studied in sun and shade leaves of several plants possessing C₃ ( Hedera helix and Laurus nobilis ) or C₄ ( Zea mays and Sorghum bicolor ) photosynthetic pathways. Sun‐shade acclimation caused marked changes in the organisation and function of photosynthetic apparatus, including significant variation in carotenoid content and composition. The contents of zanthophyll cycle pigments were higher in sun than in shade leaves in all species, but this difference was considerably greater in C₃ than in C₄ plants. The proportion of photoconvertible violaxanthin, that is the amount of violaxanthin (V) which can actually be de‐epoxidised to zeaxanthin, was much greater in sun than in shade leaves. The amount of photoconvertible V was always linearly dependent on the chlorophyll a/b ratio, although the slope of the relationship varied especially between C₃ and C₄ species. The leaf zeaxanthin and antheraxanthin contents were correlated with non‐radiative energy dissipation in all species under different light environments. These relationships were curvilinear and variable between sun and shade leaves and between C₃ and C₄ species. Hence, the dissipation of excess energy does not appear to be univocally dependent on zeaxanthin content and other photoprotective mechanisms may be involved under high irradiance stress. Such mechanisms appear largely variable between C₃ and C₄ species according to their photosynthetic characteristics.     

C4 photosynthesis in a single C3 cell is theoretically inefficient but may ameliorate internal CO2 diffusion limitations of C3 leaves

Attempts are being made to introduce C₄ photosynthetic characteristics into C₃ crop plants by genetic manipulation. This research has focused on engineering single‐celled C₄‐type CO₂ concentrating mechanisms into C₃ plants such as rice. Herein the pros and cons of such approaches are discussed with a focus on CO₂ diffusion, utilizing a mathematical model of single‐cell C₄ photosynthesis. It is shown that a high bundle sheath resistance to CO₂ diffusion is an essential feature of energy‐efficient C₄ photosynthesis. The large chloroplast surface area appressed to the intercellular airspace in C₃ leaves generates low internal resistance to CO₂ diffusion, thereby limiting the energy efficiency of a single‐cell C₄ concentrating mechanism, which relies on concentrating CO₂ within chloroplasts of C₃ leaves. Nevertheless the model demonstrates that the drop in CO₂ partial pressure, pCO₂, that exists between intercellular airspace and chloroplasts in C₃ leaves at high photosynthetic rates, can be reversed under high irradiance when energy is not limiting. The model shows that this is particularly effective at lower intercellular pCO₂. Such a system may therefore be of benefit in water‐limited conditions when stomata are closed and low intercellular pCO₂ increases photorespiration.     

Oxygen consumption during leaf nitrate assimilation in a C3 and C4 plant: the role of mitochondrial respiration

Measurements of net fluxes of CO₂ and O₂ from leaves and chlorophyll a fluorescence were used to determine the role of mitochondrial respiration during nitrate (NO₃^–) assimilation in both a C₃ (wheat) and a C₄ (maize) plant. Changes in the assimilatory quotient (net CO₂ consumed over net O₂ evolved) when the nitrogen source was shifted from NO₃^– to NH₄⁺ (ΔAQ) provided a measure of shoot NO₃^– assimilation. According to this measure, elevated CO₂ inhibited NO₃^– assimilation in wheat but not maize. Net O₂ exchange under ambient CO₂ concentrations increased in wheat plants receiving NO₃^– instead of NH₄⁺, but gross O₂ evolution from the photosynthetic apparatus (J_O2) was insensitive to nitrogen source. Therefore, O₂ consumption within wheat photosynthetic tissue (ΔΟ₂), the difference between J_O2 and net O₂ exchange, decreased during NO₃^– assimilation. In maize, NO₃^– assimilation was insensitive to changes in intercellular CO₂ concentration (C_i); nonetheless, ΔΟ₂ at low C_i values was significantly higher in NO₃^–‐fed than in NH₄⁺‐fed plants. Changes in O₂ consumption during NO₃^– assimilation may involve one or more of the following processes: (a) Mehler ascorbate peroxidase (MAP) reactions; (b) photorespiration; or (c) mitochondrial respiration. The data presented here indicates that in wheat, the last process, mitochondrial respiration, is decreased during NO₃^– assimilation. In maize, NO₃^– assimilation appears to stimulate mitochondrial respiration when photosynthetic rates are limiting.     

Optimal leaf area indices in C3 and C4 mono- and dicotyledonous species at low and high nitrogen availability

From an analytical model it was shown that for a given total amount of nitrogen in the canopy, there exists an optimal leaf area index (LAI), and therefore an optimal average leaf introgen content, at which canopy photosynthesis is maximal. If the LAI is increased above this optimum, increased light interception will not compensate for reduction in photosynthetic capacity of the canopy resulting from reduced leaf nitrogen contents. It was further derived from the model that the value of the optimal LAI increases with the photosynthetic nitrogen use efficiency (PNUE) and decreases with the canopy extinction coefficient for light (K_L) and incident photon flux density (PFD) at the top of the canopy. These hypotheses were tested on dense stands of species with different photosynthetic modes and different architectures. A garden experiment was carried out with the C₄ monocot sorghum ( Sorghum bicolor [L.] Moensch cv. Pioneer), the C₃ monocot rice ( Oryza sativa L. cv. Araure 4), the C₄ dicot amaranth ( Amaranthus cruentus L. cv. K113) and the C₃ dicot soybean ( Glycine max [L.] Merr. cv. Williams) at two levels of nitrogen availability.
The C₄ species had higher PNUEs than the C₃ species while the dicots formed stands with higher extinction coefficients for light and had lower PNUEs than the monocots. The C₄ and monocot species were found to have formed more leaf area per unit leaf nitrogen (i.e., had lower leaf nitrogen contents) than the C₃ and dicot species, respectively. These results indicate that the PNUE and the extinction coefficient for light are important factors determining the amount of leaf area produced per unit nitrogen as was predicted by the model.     

Lack of C4 photosynthetic metabolism in ears of C3 cereals

There is continuing controversy over whether a degree of C₄ photosynthetic metabolism exists in ears of C₃ cereals. In this context, CO₂ exchange and the initial products of photosynthesis were examined in flag leaf blades and various ear parts of two durum wheat (Triticum durum Desf.) and two six-rowed barley (Hordeum vulgare L.) cultivars. Three weeks after anthesis, the CO₂ compensation concentration at 210 mmol mol^?1 O₂ in durum wheat and barley ear parts was similar to or greater than that in flag leaves. The O₂ dependence of the CO₂ compensation concentration in durum wheat ear parts, as well as in the flag leaf blade, was linear, as expected for C₃ photosynthesis. In a complementary experiment, intact and attached ears and flag leaf blades of barley and durum wheat were radio-labelled with ¹⁴CO₂ during a 10s pulse, and the initial products of fixation were studied in various parts of the ears (awns, glumes, inner bracts and grains) and in the flag leaf blade. All tissues assimilated CO₂ mainly by the Calvin (C₃) cycle, with little fixation of ¹⁴CO₂ into the C₄ acids malate and aspartate (about 10% or less). These collective data support the conclusion that in the ear parts of these C₃ cereals C₄ photosynthetic metabolism is nil.     

The localization of serine hydroxymethyltransferase in leaves of C3 and C4 species

The intracellular distribution of serine hydroxymethyltransferase (EC 2.1.2.1) was studied in young wheat ( Triticum aestivum L. cv. Starke II) leaves by fractionation of protoplasts and further purification of peroxisomes and chloroplasts. Essentially all of the activity in wheat leaves was located in the mitochondria. Within the mitochondria the enzyme was mainly in the matrix as shown by centrifugation of sonicated wheat mitochondria. In the C₄ plants, Zea mays (L. cv. Earliking), Panicum miliaceum and Panicum maximum (cv. Australia) belonging to different C₄ types, serine hydroxymethyltransferase was almost exclusively found in bundle sheath cells. The location of this enzyme in leaves is consistent with its role relative to glycine decarboxylation during photorespiration.     

Differences in photosynthetic characteristics among northern and southern C4 plants

Both responses to short-term changes of temperature and to chilling under high light were analyzed in populations of Echinochloa crus-galli var. crus-galli (L.) Beauv. from Québec. North Carolina and Mississippi to improve the understanding of C₄ photosynthesis at low temperature. Comparison also included plants of Eleusine indica (L.) Gaertn. from Mississippi to provide for differences among species and populations. Plants were grown at two thermoperiods (28/22°C, 21/15°C). After transfer from cool (21/15°C) to warm (28/22°C) growth conditions, Echinochloa from Mississippi achieved the highest photosynthetic rates. Plants from Québec maintained the highest rates of CO₂ uptake upon transfer to cool conditions. Exposure to 7°C for 3 days at a photon fluence rate of 1000 μmol m−²s−¹ resulted in a reduction in the growth rates of all populations. This reduction was paralleled by a decrease in net photosynthesis and in stomatal conductance. Following chilling under hight light, the reduction in growth parameters was less important for plants from Québec than for the other populations. It suggests that, among other characteristics, northern plants had developed a certain tolerance to chilling under light.     

Seasonal water availability predicts the relative abundance of C3 and C4 grasses in Australia

Aim Numerous studies have examined the climatic factors that influence the abundance of C₄ species within the grass flora (C₄ relative species richness) in various regions throughout the world, but very few have examined the relative abundance of C₄ vs. C₃ grasses (C₄ relative abundance). We sought to determine the climatic factors that influence C₄ relative abundance throughout Australia. Location Australia (including Tasmania). Methods We measured C₄ relative abundance at 168 locations and measured δ¹³C (the abundance of ¹³C relative to ¹²C) of the bone collagen of 779 kangaroos collected throughout Australia, as bone collagen δ¹³C was assumed to be proportional to the relative abundance of C₄ grasses in the diet. Results Both C₄ relative abundance and kangaroo bone collagen δ¹³C were found to have a strong positive relationship with seasonal water availability, i.e. the distribution of rainfall in the C₄ vs. C₃ growing seasons (76% and 69% of deviance explained, respectively). There was clear evidence that seasonal water availability was a better predictor of both C₄ relative abundance and bone collagen δ¹³C than other climate variables such as mean annual temperature and January daily minimum temperature. However, seasonal water availability appeared to be a relatively poor predictor of C₄ relative species richness, which was most closely related to January daily minimum temperature (90% of deviance explained). Main conclusions Our results highlight the relatively poor relationship between C₄ relative abundance and C₄ relative species richness, and suggest that these two variables may be related to different climatic factors. They also suggest that caution is required when using C₄ relative species richness to infer the relative biomass and productivity of C₄ grasses on a global scale.