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.     

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.     

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.     

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.     

Photosynthetic responses to temperature of phosphoenolpyruvate carboxykinase type C4 species differing in cold sensitivity

Photosynthetic rates, the activities of key enzymes associated with the C₄ cycle and ribulose-1,5-bisphosphate carboxylase (RuBPCase), and the levels of metabolites involved in the C₄ cycle were compared between the two phosphoenolpyruvate carboxykinase (PCK) type C₄ species Spartina anglica, which is cold-tolerant, and Zoysia japonica, which is cold-sensitive, during exposure to low temperature. Plants of both species grown outside in summer were placed in a growth chamber at 27/20 °C day/night temperatures. After 1 week, plants were exposed to 20/17 °C for 1 week and then to 10/7 °C for 2 weeks. Photosynthetic rates in Z. japonica decreased progressively to about 25% during the chilling treatments. In contrast, S. anglica exhibited a 43% increase in photosynthetic rates after exposure to 20 °C for 1 week, which remained relatively constant thereafter. Consistent with these observations, most of the C₄ enzymes and RuBPCase in Z. japonica declined. Phosphoenolpyruvate carboxylase (PEPC) and PCK activities declined particularly drastically during the treatments. However, the activities of these enzymes in S. anglica showed either a slight increase or decrease upon a mild cold treatment, and remained relatively constant during further chilling treatments. There was a sharp decline in phosphoenolpyruvate in Z. japonica after exposure to 10 °C. On the other hand, metabolite levels in S. anglica were largely unaffected by the chilling treatments. These results suggest that the drastic declines of both PEPC and PCK activities may be important limiting factors responsible for cold sensitivity in C₄ photosynthesis of Z. japonica.     

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.     

Photosynthesis, immediate export and carbon partitioning in source leaves of C3, C3-C4 intermediate, and C4Panicum and Flaveria species at ambient and elevated CO2 levels

Immediate export in leaves of C₃‐C₄ intermediates were compared with their C₃ and C₄ relatives within the Panicum and Flaveria genera. At 35 Pa CO₂, photosynthesis and export were highest in C₄ species in each genera. Within the Panicum, photosynthesis and export in ‘type I’ C₃‐C₄ intermediates were greater than those in C₃ species. However, ‘type I’ C₃‐C₄ intermediates exported a similar proportion of newly fixed ¹⁴C as did C₄ species. Within the Flaveria, ‘type II’ C₃‐C₄ intermediate species had the lowest export rather than the C₃ species. At ambient CO₂, immediate export was strongly correlated with photosynthesis. However, at 90 Pa CO₂, when photosynthesis and immediate export increased in all C₃ and C₃‐C₄ intermediate species, proportionally less C was exported in all photosynthetic types than that at ambient CO₂. All species accumulated starch and sugars at both CO₂ levels. There was no correlation between immediate export and the pattern of ¹⁴C‐labelling into sugars and starch among the photosynthetic types within each genus. However, during CO₂ enrichment, C₄Panicum species accumulated sugars above the level of sugars and starch normally made at ambient CO₂, whereas the C₄Flaveria species accumulated only additional starch.     

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.     

C4 photosynthesis in Cyperus longus L., a species occurring in temperate climates

Abstract. Cyperus longus L. , which has a widespread but disjunct distribution throughout Europe and extends northwards into Britain, was found to be a C₄ species based upon its Kranz leaf anatomy, low CO₂ compensation point and the labelling of malate as an early product of ¹⁴CO₂ fixation. The photosynthetic characteristics of C. longus are similar to many other C₄ species with a high maximum rate of photosynthesis (> 1.5 mg CO₂ m ⁻² s ⁻¹) and a relatively high temperature optimum (30–35°C), but unlike many C₄ species the rate of photosynthesis does not decline rapidly below the optimum temperature and a substantial rate (0.6 mgCO₂ m⁻²s⁻¹)occursat 15°C. Leaf extension is very slow at 15°C and shows a curvilinear response to temperatures between 15 and 25°C. Leaves extend at a rate of almost 4 cm d⁻¹ at 25°C.     

Climate controls on C3 vs. C4 productivity in North American grasslands from carbon isotope composition of soil organic matter

We analyzed the δ¹³C of soil organic matter (SOM) and fine roots from 55 native grassland sites widely distributed across the US and Canadian Great Plains to examine the relative production of C₃ vs. C₄ plants (hereafter %C₄) at the continental scale. Our climate vs. %C₄ results agreed well with North American field studies on %C₄, but showed bias with respect to %C₄ from a US vegetation database (statsgo ) and weak agreement with a physiologically based prediction that depends on crossover temperature. Although monthly average temperatures have been used in many studies to predict %C₄, our analysis shows that high temperatures are better predictors of %C₄. In particular, we found that July climate (average of daily high temperature and month's total rainfall) predicted %C₄ better than other months, seasons or annual averages, suggesting that the outcome of competition between C₃ and C₄ plants in North American grasslands was particularly sensitive to climate during this narrow window of time. Root δ¹³C increased about 1‰ between the A and B horizon, suggesting that C₄ roots become relatively more common than C₃ roots with depth. These differences in depth distribution likely contribute to the isotopic enrichment with depth in SOM where both C₃ and C₄ grasses are present.     

Bienertia cycloptera Bunge ex Boiss., Chenopodiaceae, another C4 Plant without Kranz Tissues286

Abstract: Following up an earlier study on Borszczowia aralocaspica, a second C₄ leaf type without Kranz tissues is described from Bienertia cycloptera Bunge ex Boiss. The species belongs also to tribe Suaedeae and grows in similar temporarily wet saline habitats in southwestern C Asia. Like Borszczowia, the species has the ability to perform C₄ photosynthesis in single chlorenchyma cells but the cytological compartmentation differs. Evidence is cited from anatomy (cytoplasmic compartmentation, starch formation), from carbon isotope composition and from indirect sources. It is documented by micrographs and δ¹³C values (mean ‐ 14.7 %). Variations of the δ¹³C values subsequently found in newly formed leaves of the same greenhouse plants (‐ 15.5 to ‐ 21.1 %) support the hypothesis that Bienertia is a facultative C₄/C₃ species. The new described bienertioid leaf type differs from the leaves of C₃ species in Suaeda by strict separation of the mesophyll into a central aqueous tissue without chloroplasts and a peripheral 1 ‐ 3 layered chlorenchyma with elaborate cytoplasmic compartmentation. Each cell of the latter contains a peripheral cytoplasmic layer with scattered chloroplasts and a large globular cytoplasmic body located in the central vacuole which is densely packed with starch producing chloroplasts, and joined by the nucleus. Comparative studies on different leaves of the same individuals of ordinary C₄ species demonstrated a remarkable range of variation, from 1.0 % in Salsola tragus up to 2.5 % in Petrosimonia sibirica. This corresponds well with the variance in data from different individuals and populations. For explaining the larger interspecific variation from about 23 other species of Salsoloideae, the hypothesis is developed that in species or groups of taxa specific leakages of their C₄ systems do occur. By three characters unique in tribe Suaedeae and detected during the study, Bienertia is confirmed as well separated from Suaeda and as a monotypic genus.     

Elevated CO2 enhances water relations and productivity and affects gas exchange in C3 and C4 grasses of the Colorado shortgrass steppe.

Six open‐top chambers were installed on the shortgrass steppe in north‐eastern Colorado, USA from late March until mid‐October in 1997 and 1998 to evaluate how this grassland will be affected by rising atmospheric CO₂. Three chambers were maintained at current CO₂ concentration (ambient treatment), three at twice ambient CO₂, or approximately 720 μmol mol^?1 (elevated treatment), and three nonchambered plots served as controls. Above‐ground phytomass was measured in summer and autumn during each growing season, soil water was monitored weekly, and leaf photosynthesis, conductance and water potential were measured periodically on important C₃ and C₄ grasses. Mid‐season and seasonal above‐ground productivity were enhanced from 26 to 47% at elevated CO₂, with no differences in the relative responses of C₃/C₄ grasses or forbs. Annual above‐ground phytomass accrual was greater on plots which were defoliated once in mid‐summer compared to plots which were not defoliated during the growing season, but there was no interactive effect of defoliation and CO₂ on growth. Leaf photosynthesis was often greater in Pascopyrum smithii (C₃) and Bouteloua gracilis (C₄) plants in the elevated chambers, due in large part to higher soil water contents and leaf water potentials. Persistent downward photosynthetic acclimation in P. smithii leaves prevented large photosynthetic enhancement for elevated CO₂‐grown plants. Shoot N concentrations tended to be lower in grasses under elevated CO₂, but only Stipa comata (C₃) plants exhibited significant reductions in N under elevated compared to ambient CO₂ chambers. Despite chamber warming of 2.6 °C and apparent drier chamber conditions compared to unchambered controls, above‐ground production in all chambers was always greater than in unchambered plots. Collectively, these results suggest increased productivity of the shortgrass steppe in future warmer, CO₂ enriched environments.     

Utilization of O2 in the metabolic optimization of C4 photosynthesis

The combined effects of O₂ on net rates of photosynthesis, photosystem II activity, steady‐state pool size of key metabolites of photosynthetic metabolism in the C₄ pathway, C₃ pathway and C₂ photorespiratory cycle and on growth were evaluated in the C₄ species Amaranthus edulis and the C₃ species Flaveria pringlei. Increasing O₂ reduced net CO₂ assimilation in F. pringlei due to an increased flux of C through the photorespiratory pathway. However, in A. edulis increasing O₂ up to 5–10% stimulated photosynthesis. Analysis of the pool size of key metabolites in A. edulis suggests that while there is some O₂ dependent photorespiration, O₂ is required for maximizing C₄ cycle activity to concentrate CO₂ in bundle sheath cells. Therefore, the response of net photosynthesis to O₂ in C₄ plants may result from the balance of these two opposing effects. Under 21 versus 5% O₂, growth of A. edulis was stimulated about 30% whereas that of F. pringlei was inhibited about 40%.     

The growth response of C4 plants to rising atmospheric CO2 partial pressure: a reassessment

Despite mounting evidence showing that C₄ plants can accumulate more biomass at elevated CO₂ partial pressure (p(CO₂)), the underlying mechanisms of this response are still largely unclear. In this paper, we review the current state of knowledge regarding the response of C₄ plants to elevated p(CO₂) and discuss the likely mechanisms. We identify two main routes through which elevated p(CO₂) can stimulate the growth of both well-watered and water-stressed C₄ plants. First, through enhanced leaf CO₂ assimilation rates due to increased intercellular p(CO₂). Second, through reduced stomatal conductance and subsequently leaf transpiration rates. Reduced transpiration rates can stimulate leaf CO₂ assimilation and growth rates by conserving soil water, improving shoot water relations and increasing leaf temperature. We argue that bundle sheath leakiness, direct CO₂ fixation in the bundle sheath or the presence of C₃-like photosynthesis in young C₄ leaves are unlikely explanations for the high CO₂-responsiveness of C₄ photosynthesis. The interactions between elevated p(CO₂), leaf temperature and shoot water relations on the growth and photosynthesis of C₄ plants are identified as key areas needing urgent research.     

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.     

A critical appraisal of a combined stomatal-photosynthesis model for C3 plants

Gas-exchange measurements on Eucalyptus grandis leaves and data extracted from the literature were used to test a semi-empirical model of stomatal conductance for CO₂ g_Sc=g_o+a₁A/(c_s-I) (1+D_s/D_o)] where A is the assimilation rate; D_s and c_s are the humidity deficit and the CO₂ concentration at the leaf surface, respectively; g₀ is the conductance as A → 0 when leaf irradiance → 0; and D₀ and a₁ are empirical coefficients. This model is a modified version of g_sc=a₁A h_s/c_s first proposed by Ball, Woodrow & Berry (1987, in Progress in Photosynthesis Research, Martinus Mijhoff, Publ., pp. 221–224), in which h_s is relative humidity. Inclusion of the CO₂ compensation point, τ, improved the behaviour of the model at low values of c_s, while a hyperbolic function of D_s for humidity response correctly accounted for the observed hyperbolic and linear variation of g_sc and c_i/c_s as a function of D_s, where C_i is the intercellular CO₂ concentration. In contrast, use of relative humidity as the humidity variable led to predictions of a linear decrease in g_sc and a hyperbolic variation in c_i/c_s as a function of D_s, contrary to data from E. grandis leaves. The revised model also successfully described the response of stomata to variations in A, D_s and c_s for published responses of the leaves of several other species. Coupling of the revised stomatal model with a biochemical model for photosynthesis of C₃ plants synthesizes many of the observed responses of leaves to light, humidity deficit, leaf temperature and CO₂ concentration. Best results are obtained for well-watered 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.     

Insects and fungi on a C3 sedge and a C4 grass exposed to elevated atmospheric CO2 concentrations in open-top chambers in the field

The effects of elevated atmospheric CO₂ concentration on plant-fungi and plant-insect interactions were studied in an emergent marsh in the Chesapeake Bay. Stands of the C₃ sedge Scirpus olneyi Grey, and the C₄ grass Spartina patens (Ait.) Muhl. have been exposed to elevated atmospheric CO₂ concentrations during each growing season since 1987. In August 1991 the severities of fungal infections and insect infestations were quantified. Shoot nitrogen concentration ([N]) and water content (WC) were determined. In elevated concentrations of atmospheric CO₂, 32% fewer S. olneyi plants were infested by insects, and there was a 37% reduction in the severity of a pathogenic fungal infection, compared with plants grown in ambient CO₂ concentrations. S. olneyi also had reduced [N], which correlated positively with the severities of fungal infections and insect infestations. Conversely, S. patens had increased WC but unchanged [N] in elevated concentrations of atmospheric CO₂ and the severity of fungal infection increased. Elevated atmospheric CO₂ concentration increased or decreased the severity of fungal infection depending on at least two interacting factors, [N] and WC; but it did not change the number of plants that were infected with fungi. In contrast, the major results for insects were that the number of plants infected with insects decreased, and that the amount of tissue that each insect ate also decreased.