C3 grasses have higher nutritional quality than C4 grasses under ambient and elevated atmospheric CO2

Grasses with the C₃ photosynthetic pathway are commonly considered to be more nutritious host plants than C₄ grasses, but the nutritional quality of C₃ grasses is also more greatly impacted by elevated atmospheric CO₂ than is that of C₄ grasses; C₃ grasses produce greater amounts of nonstructural carbohydrates and have greater declines in their nitrogen content than do C₄ grasses under elevated CO₂. Will C₃ grasses remain nutritionally superior to C₄ grasses under elevated CO₂ levels? We addressed this question by determining whether levels of protein in C₃ grasses decline to similar levels as in C₄ grasses, and whether total carbohydrate : protein ratios become similar in C₃ and C₄ grasses under elevated CO₂. In addition, we tested the hypothesis that, among the nonstructural carbohydrates in C₃ grasses, levels of fructan respond most strongly to elevated CO₂. Five C₃ and five C₄ grass species were grown from seed in outdoor open‐top chambers at ambient (370 ppm) or elevated (740 ppm) CO₂ for 2 months. As expected, a significant increase in sugars, starch and fructan in the C₃ grasses under elevated CO₂ was associated with a significant reduction in their protein levels, while protein levels in most C₄ grasses were little affected by elevated CO₂. However, this differential response of the two types of grasses was insufficient to reduce protein in C₃ grasses to the levels in C₄ grasses. Although levels of fructan in the C₃ grasses tripled under elevated CO₂, the amounts produced remained relatively low, both in absolute terms and as a fraction of the total nonstructural carbohydrates in the C₃ grasses. We conclude that C₃ grasses will generally remain more nutritious than C₄ grasses at elevated CO₂ concentrations, having higher levels of protein, nonstructural carbohydrates, and water, but lower levels of fiber and toughness, and lower total carbohydrate : protein ratios than C₄ grasses.     

Distribution of C3 and C4 grasses along an altitudinal gradient in Central Argentina

The distribution pattern of C₃ and C₄ grasses was studied in eight sites located between 350 m and 2100 m along an altitudinal gradient in Central Argentina. Of 139 taxa fifty-nine are C₃ and eighty C₄. Species of the C₃ tribes (Stipeae, Poeae, Meliceae, Aveneae, Bromeae and Triticeae) and C₃ Paniceae species increase in number at higher elevations; only one C₃ species was found below 650 m. C₄ Aristideae, Pappophoreae, Eragrostideae, Cynodonteae, Andropogoneae and Paniceae increase at lower altitudes. The floristic crossover point is at about 1500 m; the ground cover cross-over point is at about 1000 m. Analysis of the relationships between % C₄ species along the gradient and nine climatic and environmental variables showed the highest correlation with July mean temperature, but all temperature variables show highly significant correlations with % C₄. Correlation with annual rainfall is lower but also significant. These results are consistent with previous research showing the relative importance of C₄ grasses as temperature increases. C₃ species make a high contribution to relative grass coverage below the C₃/C₄ floristic crossover point but are rare below 1000 m.     

Photosynthetic pathway variation among C4 grasses along a precipitation gradient in Argentina

Aim Based on the biochemical and physiological attributes of C₄ grasses, and on the close association between decarboxylation pathways and the taxa in which they evolved, the hypotheses tested were: (1) that C₄ grasses would become progressively more abundant as precipitation decreased, with grasses of the NADP‐me subtype more abundant in wetter sites and those of the NAD‐me subtype more common in arid regions; and (2) that the distribution of grass subfamilies would also be correlated with annual precipitation. Location The study was conducted along a precipitation gradient in central Argentina, from the eastern Pampas (>1000 mm year^?1) to the western deserts and semi‐deserts near the Andes (<100 mm year^?1). Methods Percentage of species and relative cover of C₃ and C₄ grasses (including C₄ subtypes) in local floras from 15 lowland sites of central Argentina were obtained from our own unpublished data and from recently published floristic surveys. Pearson correlation coefficients were obtained between grass distribution parameters and the available climatic data. Results The percentage of C₄ grasses increased towards the arid extreme and showed a strong negative correlation with annual rainfall (r = ?0.74, P < 0.01). Within the C₄ subtypes, the NADP‐me species showed a higher proportional representation at the wetter extreme, whereas the representation of NAD‐me species increased towards the more arid extreme. The relationship of PEP‐ck species with climatic parameters in central Argentina was less evident. The distributions of the Panicoideae and Chloridoideae subfamilies along the precipitation gradient were diametrically opposed, with the Panicoideae positively (r = 0.86, P < 0.001) and the Chloridoideae negatively (r = ?0.87, P < 0.001) correlated with annual precipitation. Main conclusions Our data are consistent with the broad observation that C₄ grasses tend to dominate in areas where the wet season falls in the warmer summer months. In agreement with previously reported results for Africa, Asia, Australia and North America, we describe here for the first time a significant relationship between annual precipitation and the prevalence of the NADP‐me and NAD‐me photosynthetic pathways along climatic gradients for the Neotropics. We also report for the first time that correlations between C₄ species and annual rainfall are stronger when the relative cover of grass species is considered. The association of grass subfamilies Panicoideae and Chloridoideae with rainfall is as strong as that recorded for the NADP‐me and NAD‐me variants, respectively, suggesting that characteristics other than decarboxylation type may be responsible for the geographic patterns described in this study.     

The photosynthesis of young Panicum C4 leaves is not C3-like

Evidence is presented contrary to the suggestion that C₄ plants grow larger at elevated CO₂ because the C₄ pathway of young C₄ leaves has C₃-like characteristics, making their photosynthesis O₂ sensitive and responsive to high CO₂. We combined PAM fluorescence with gas exchange measurements to examine the O₂ dependence of photosynthesis in young and mature leaves of Panicum antidotale (C₄, NADP-ME) and P. coloratum (C₄, NAD-ME), at an intercellular CO₂ concentration of 5 Pa. P. laxum (C₃) was used for comparison. The young C₄ leaves had CO₂ and light response curves typical of C₄ photosynthesis. When the O₂ concentration was gradually increased between 2 and 40%, CO₂ assimilation rates (A) of both mature and young C₄ leaves were little affected, while the ratio of the quantum yield of photosystem II to that of CO₂ assimilation (Φ_PSII/Φ_CO2) increased more in young (up to 31%) than mature (up to 10%) C₄ leaves. A of C₃ leaves decreased by 1·3 and Φ_PSII/Φ_CO2 increased by 9-fold, over the same range of O₂ concentrations. Larger increases in electron transport requirements in young, relative to mature, C₄ leaves at low CO₂ are indicative of greater O₂ sensitivity of photorespiration. Photosynthesis modelling showed that young C₄ leaves have lower bundle sheath CO₂ concentration, brought about by higher bundle sheath conductance relative to the activity of the C₄ and C₃ cycles and/or lower ratio of activities of the C₄ to C₃ cycles.     

The origins and diversification of C4 grasses and savanna-adapted ungulates

C₄ grasses constitute the main component of savannas and are pervasive in other dry tropical ecosystems where they serve as the main diet for grazing animals. Among potential factors driving C₄ evolution of grasses, the interaction between grasses and grazers has not been investigated. To evaluate if increased grazing pressure may have selected for higher leaf silica production as the grasses diverged, we reconstructed the phylogeny of all 800 genera of the grass family with both molecular (combined multiplastid DNA regions) and morphological characters. Using molecular clocks, we also calculated the age and number of origins of C₄ clades and found that shifts from C₃ to C₄ photosynthesis occurred at least 12 times starting 30.9 million years ago and found evidence that the most severe drop in atmospheric carbon dioxide in the late Oligocene (between 33 and 30 million years ago) matches the first origin of C₄ photosynthesis in Chloridoideae. By combining fossil and phylogenetic data for ungulates and implementing a randomization procedure, our results showed that the appearance of C₄ grass clades and ungulate adaptations to C₄-dominated habitats match significantly in time. An increase of leaf epidermal density of silica bodies was found to correspond to postulated shifts in diversification rates in the late Miocene [24 significant shifts in diversification ( P <0.05) were detected between 23 and 3.7 million years ago]. For aristidoid and chloridoid grasses, increased grazing pressure may have selected for a higher leaf epidermal silica production in the late Miocene.     

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.     

Ancient latitudinal gradients of C3/C4 grasses interpreted from stable isotopes of New World Pleistocene horse (Equus) teeth

Carbon and oxygen isotopic data are reported from 116 Pleistocene Equus teeth from sixty-six localities in the New World ranging from 68°N (Alaska, Canada) to 35°S (Argentina). Equus species have been predominantly grazers, and as such, carbon isotopic values of their tooth enamel provide evidence of the Pleistocene distribution of C₃ and C₄ grasses. The carbon data presented here indicate a gradient (δ¹³C range of 10 parts/mil) in the relative proportion of C₃ and C₄ grasses between high latitude and equatorial Equus samples. The largest amount of change from C₃ to C₄ grasses during the Pleistocene occurred in the mid-latitudes between about 30 to 40°. The oxygen data, which vary proportionately with temperature, indicate a latitudinal gradient (δ¹⁸O range of 20 parts/mil) between high-latitude and equatorial Equus samples. The basic pattern of latitudinal gradients of C₃/C₄ grass distribution and temperature as interpreted from these Pleistocene data is similar to the modern-day. The use of stable isotopes of fossil herbivore teeth represents a new means to interpret Pleistocene climates and terrestrial ecology.     

Comparative ultrastructure and gas exchange characteristics of the C3–C4 intermediate Neurachne minor S. T. Blake (Poaceae)

Abstract Ultrastructural and physiological characteristics of the C₃-C₄ intermediate Neurachne minor S. T. Blake (Poaceae) are compared with those of C₃ and C₄ relatives, and C₃-C₄Panicum milioides Nees ex Trin. N. minor consistently exhibits very low CO₂ compensation points (τ: 1.0, usually 0.3–0.6 Pa) yet has C₃-like δ¹³C values. CO₂ assimilation rates (A) respond like those of C₃ plants to a decrease in O₂ partial pressure (2 × 104–1.9 × 103 Pa) at ambient CO₂ levels, but this response is progressively attenuated until negligible at very low CO₂. By contrast, other species of the Neurachneae are clearly either C₄ (two spp.) or C₃ (seven spp.). For plants grown and measured at different photon flux densities (PFDs), τ for N. minor and P. milioides increases from 0.5 to 1.0, and from 1.0 to 2.1 Pa, respectively, as PFD is decreased from 1860 to 460 μmol m^?2s^?1. In N. minor, the O₂ response of τ is either biphasic as in P. milioides, but much diminished and with a higher transition point, or is very C₄-like. As in C₄ relatives, inner sheath cells contain numerous chloroplasts. Their walls possess a suberized lamella, which may make them more CO₂-tight than bundle sheath cells of P. milioides, contributing to the almost C₄-like τ characteristics of N. minor. The biochemical basis of C₃-C₄ intermediacy is considered.     

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.     

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.     

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.     

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.     

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.     

Different Tolerance to Light Stress in NO3−- and NH4+-Grown Phaseolus vulgaris L.

Abstract: NH₄⁺‐grown plants are more sensitive to light stress than NO₃^?‐grown plants, as indicated by reduced growth and intervenal chlorosis of French bean (Phaseolus vulgaris L.). Measuring the time course of F_v/F_m ratios under photoinhibitory light regimes did not reveal any difference in PS II damage between NO₃^?‐ and NH₄⁺‐grown plants, in spite of some indications of higher energy quenching in NO₃^?‐grown plants. Also, a direct action of NH₄⁺ as an uncoupler at the thylakoid membrane could be excluded. Instead, biochemical analysis revealed enhanced lipid peroxidation and higher activity of scavenging enzymes in NH₄⁺‐grown plants indicating that these plants make use of metabolic pathways with stronger radical formation. Evidence for higher rates of photorespiration in NH₄⁺‐grown plants came from experiments showing that electron flux and O₂ evolution were decreased by SHAM in NH₄⁺‐grown plants, and by antimycin A in NO₃^?‐grown plants. Further, the comparison of electron flux and of photoacoustic measurements of O₂ evolution suggested that in NH₄⁺‐grown plants the Mehler reaction was also increased, at least in the induction phase. However, the major cause of N form‐dependent stress sensitivity is assumed to be in the coupling between photosynthesis and respiration, i.e., NO₃^?‐grown plants can utilize the TCA cycle for the generation of C skeletons for amino acid synthesis, thus improving the ATP: reductant balance, whereas NH₄⁺‐grown plants have enhanced rates of photorespiration.     

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.     

C4 photosynthesis at low temperatures

Abstract. C₄ plants grown in optimum conditions are, by comparison to C₃, capable of higher maximum dry-matter yields and greater efficiencies of water and nitrogen use, yet they are rare outside the subtropics. Both latitudinal and altitudinal limits of C₄ distributions correlate most closely with a mean minimum temperature of 8-10°C during the period of active growth. The possibility that the C₄ process is inherently incapable of functioning at low temperatures is examined. The reversible effects of chilling on the quantum efficiency of C₄ photosynthesis and the functioning of the individual steps in the C₄ cycle are examined. Chilling also produces an irreversible loss of capacity to assimilate CO₂ which is directly proportional to the light received during chilling. It is suggested that the reversible reduction in capacity to assimilate CO₂ and the lack of an alternative pathway for the utilization of lightgenerated reducing power may make C₄ species more prone to chilling-dependent photoinhibition. Laboratory studies and limited field observations suggest that this damage would be most likely to occur during photosynthetic induction at the temperatures and light levels encountered on clear, cool mornings during the spring and early summer in cool climates. Even those C₄ species occurring naturally in cool climates do not appear fully capable of tolerating these conditions; indeed their growth patterns suggest that they may be adapted by avoiding 'rather than enduring' such conditions.