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.     

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.     

Carbonic anhydrase and C4 photosynthesis: a transgenic analysis

Carbonic anhydrase (CA, EC 4.2.1.1) catalyses the first reaction in the C₄ photosynthetic pathway, the conversion of atmospheric CO₂ to bicarbonate in the mesophyll cytosol. To examine the importance of the enzyme to the functioning of the C₄ photosynthetic pathway, Flaveria bidentis (L.) Kuntze, a C₄ dicot, was genetically transformed with an antisense construct in which the cDNA encoding a putative cytosolic CA (CA3) was placed under the control of a constitutive promoter. Some of the primary transformants had impaired CO₂ assimilation rates and required high CO₂ for growth. The T₁ progeny of four primary transformants were used to examine the quantitative relationship between leaf CA activity and CO₂ assimilation rate. CA activity was determined in leaf extracts with a mass spectrometric technique that measured the rate of ¹⁸O exchange from doubly labelled ¹³C¹⁸O₂. Steady‐state CO₂ assimilation rates were unaffected by a decrease in CA activity until CA activity was less than 20% of wild type when they decreased steeply. Transformants with less than 10% of wild‐type CA activity had very low CO₂ assimilation rates and grew poorly at ambient CO₂ partial pressure. Reduction in CA activity also increased the CO₂ partial pressure required to saturate CO₂ assimilation rates. The present data show that CA activity is essential for the functioning of the C₄ photosynthetic pathway.     

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.     

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%.     

Effect of nitrogen nutrition on photosynthesis and growth in C4Panicum species

Abstract. The growth and photosynthetic responses to high and low N nutrition were measured in 2 NADP-malic enzyme and 4 NAD-malic enzyme C₄ subtype Panicum species to evaluate whether differences in C₄ photosynthetic biochemistry result in differences in the N requirement for growth. All species had lower biomass production, photosynthesis rates, and shoot N concentrations at low N, and no consistent differences between the C₄ subtypes were apparent. The assimilation rates (biomass accumulated over the period of growth) for the NADP-malic enzyme species were higher than the NAD-malic enzyme species at high N but not at low N. When assimilation rates were evaluated on a shoot N basis a higher N-use-efficiency was found for the NADP-malic enzyme species at high N. Thus the NADP-malic enzyme Panicum species had a greater amount of growth for a given shoot N concentration, but only above a certain level of shoot N concentrations.     

Oxygen sensitivity of C4 photosynthesis: evidence from gas exchange and chlorophyll fluorescence analyses with different C4 subtypes

Because photosynthetic rates in C₄ plants are the same at normal levels of O₂ (c, 20 kPa) and at c, 2 kPa O₂ (a conventional test for evaluating photorespiration in C₃ plants) it has been thought that C₄ photosynthesis is O₂ insensitive. However, we have found a dual effect of O₂ on the net rate of CO₂ assimilation among species representing all three C₄ subtypes from both monocots and dicots. The optimum O₂ partial pressure for C₄ photosynthesis at 30 °C, atmospheric CO₂ level, and half full sunlight (1000 μmol quanta m^?2 s^?1) was about 5–10 kPa. Photosynthesis was inhibited by O₂ below or above the optimum partial pressure. Decreasing CO₂ levels from ambient levels (32.6 Pa) to 9.3 Pa caused a substantial increase in the degree of inhibition of photosynthesis by supra-optimum levels of O₂ and a large decrease in the ratio of quantum yield of CO₂ fixation/quantum yield of photosystem II (PSII) measured by chlorophyll a fluorescence. Photosystem II activity, measured from chlorophyll a fluorescence analysis, was not inhibited at levels of O₂ that were above the optimum for CO₂ assimilation, which is consistent with a compensating, alternative electron How as net CO₂ assimilation is inhibited. At suboptimum levels of O₂, however, the inhibition of photosynthesis was paralleled by an inhibition of PSII quantum yield, increased state of reduction of quinone A, and decreased efficiency of open PSII centres. These results with different C₄ types suggest that inhibition of net CO₂ assimilation with increasing O₂ partial pressure above the optimum is associated with photorespiration, and that inhibition below the optimum O₂ may be caused by a reduced supply of ATP to the C₄ cycle as a result of inhibition of its production photochemically.     

An examination of the advantages of C3-C4 intermediate photosynthesis in warm environments

Abstract. The photosynthetic responses to temperature in C₃, C₃-C₄ intermediate, and C₄ species in the genus Flaveria were examined in an effort to identify whether the reduced photorespiration rates characteristic of C₃-C₄ intermediate photosynthesis result in adaptive advantages at warm leaf temperatures. Reduced photorespiration rates were reflected in lower CO₂ compensation points at all temperatures examined in the C₃-C₄ intermediate, Flaveria floridana, compared to the C₃ species, F. cronquistii. The C₃-C₄ intermediate, F. floridana, exhibited a C₃-like photosynthetic temperature dependence, except for relatively higher photosynthesis rates at warm leaf temperatures compared to the C₃ species, F. cronquistii. Using models of C₃ and C₃-C₄ intermediate photosynthesis, it was predicted that by recycling photorespired CO₂ in bundle-sheath cells, as occurs in many C₃-C₄ intermediates, photosynthesis rates at 35°C could be increased by 28%, compared to a C₃ plant. Without recycling photorespired CO₂, it was calculated that in order to improve photosynthesis rates at 35°C by this amount in C₃ plants, (1) intercellular CO₂ partial pressures would have to be increased from 25 to 31 Pa, resulting in a 57% decrease in water-use efficiency, or (2) the activity of RuBP carboxylase would have to be increased by 32%, resulting in a 22% decrease in nitrogen-use efficiency. In addition to the recycling of photorespired CO₂, leaves of F. floridana appear to effectively concentrate CO₂ at the active site of RuBP carboxylase, increasing the apparent carboxylation efficiency per unit of in vitro RuBP carboxylase activity. The CO₂-concentrating activity also appears to reduce the temperature sensitivity of the carboxylation efficiency in F. floridana compared to F. cronquistii. The carboxylation efficiency per unit of RuBP carboxylase activity decreased by only 38% in F. floridana, compared to 50% in F. cronquistii, as leaf temperature was raised from 25 to 35°C. The C₃-C₄ intermediate, F. ramosissima, exhibited a photosynthetic temperature temperature response curve that was more similar to the C₄ species, F. trinervia, than the C₃ species, F. cronquistii. The C₄-like pattern is probably related to the advanced nature of C₄-like biochemical traits in F. ramosissima The results demonstrate that reductions in photorespiration rates in C₃-C₄ intermediate plants create photosynthetic advantages at warm leaf temperatures that in C₃ plants could only be achieved through substantial costs to water-use efficiency and/or nitrogen-use efficiency.     

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.     

Stomatal mechanism as the basis of the evolution of CAM and C4 photosynthesis

Abstract. The similarities between the component reactions of the presently known variants of photosynthetic carbon metabolism (crassulacean acid metabolism, the acid metabolism of Tillandsia usneoides , aquatic acid metabolism, and C₄ photosynthesis) when considered along with their widely scattered taxonomic distribution strongly suggest polyphyletic origins resulting from evolutionary modification of a common, universally distributed metabolic sequence. The synthesis and consumption of four-carbon acids in the cation-balancing reactions involved in the regulation of stomatal aperture appear to exhibit all of the characteristics likely to be displayed by such a metabolic progenitor.
The present status of the proposal that the expression of aspects of stomatal metabolism in photosynthetic mesophyll cells represents the basis for the evolution of the variant of photosynthetic carbon metabolism is discussed. The prospects of experimental approaches which may yield information relevant to the proposal are also explored.     

The evolutionary relationship between stomatal mechanism, crassulacean acid metabolism and C4 photosynthesis

Abstract. All of the features of crassulacean acid metabolism (CAM) and most characteristics of C₄ photosynthesis are exhibited by stomatal guard cells. It is proposed that CAM and possibly also C₄ photosynthesis result from the expression in photosynthetic cells of genetic information which is expressed only in guard cells of C₃ plants.     

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.     

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).     

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.     

Environmental and Evolutionary Preconditionsfor the Origin and Diversification of the C4 PhotosyntheticSyndrome

Abstract: C₄ photosynthesis is an evolutionary solution to high rates of photorespiration and low kinetic efficiency of Rubisco in CO₂‐depleted atmospheres of recent geologic time. About 7500 plant species are C₄, in contrast to 30 000 CAM and 250 000 C₃ species. All C₄ plants occur in approximately 90 genera from 18 angiosperm families. In all of these families, the C₄ pathway evolved independently. In many, multiple independent origins have occurred, such that over 30 distinct evolutionary origins of the C₄ pathway are recognized. Fossil and carbon isotope evidence show that the C₄ syndrome is at least 12 to 15 million years old, although estimates based on molecular sequence comparisons indicate it is over 20 million years old. The evolutionary radiation of herbaceous angiosperms may have been required for C₄ plant evolution. All C₄ species occur in advanced angiosperm families that appeared in the fossil record in the past 70 million years. Most of these families diversified in terms of genera and species numbers between 20 to 40 million years ago, during a period of global cooling, atmospheric CO₂ reduction and aridification. During the period of diversification, numerous traits arose in the C₃ flora that enhanced their performance in arid environments and atmospheres of reduced CO₂. Some of these traits may have predisposed certain taxa to develop the C₄ pathway once atmospheric CO₂ levels declined to a point where the ability to concentrate CO₂ had a selective advantage. Leading traits in C₃ plants that may have facilitated the initial transition to C₄ photosynthesis include close vein spacing and an enlargement of the bundle sheath cell layer to form a Kranz‐like anatomy. Ecological factors not directly connected with photosynthesis probably also played a role. For example, extensive ecological disturbance may have been needed to convert C₃‐dominated woodlands into open, high‐light habitats where herbaceous C₄ plants could succeed. Disturbances in the form of fire, and browsing by large mammals, increase during the time of C₄ plant evolution and diversification. Fire increased because of the drying climate, while browsing increased with the evolutionary diversification of the mammalian megafauna in the Oligocene and Miocene epochs. In summary, the origin of C₄ plants is hypothesized to have resulted from a novel combination of environmental and phylogenetic developments that, for the first time, established the preconditions required for C₄ plant evolution.     

Low-temperature photosynthetic performance of a C4 grass and a co-occurring C3 grass native to high latitudes

The photosynthetic performance of C₄ plants is generally inferior to that of C₃ species at low temperatures, but the reasons for this are unclear. The present study investigated the hypothesis that the capacity of Rubisco, which largely reflects Rubisco content, limits C₄ photosynthesis at suboptimal temperatures. Photosynthetic gas exchange, chlorophyll a fluorescence, and the in vitro activity of Rubisco between 5 and 35 °C were measured to examine the nature of the low‐temperature photosynthetic performance of the co‐occurring high latitude grasses, Muhlenbergia glomerata (C₄) and Calamogrostis canadensis (C₃). Plants were grown under cool (14/10 °C) and warm (26/22 °C) temperature regimes to examine whether acclimation to cool temperature alters patterns of photosynthetic limitation. Low‐temperature acclimation reduced photosynthetic rates in both species. The catalytic site concentration of Rubisco was approximately 5.0 and 20 µmol m^?2 in M. glomerata and C. canadensis, respectively, regardless of growth temperature. In both species, in vivo electron transport rates below the thermal optimum exceeded what was necessary to support photosynthesis. In warm‐grown C. canadensis, the photosynthesis rate below 15 °C was unaffected by a 90% reduction in O₂ content, indicating photosynthetic capacity was limited by the capacity of P_i‐regeneration. By contrast, the rate of photosynthesis in C. canadensis plants grown at the cooler temperatures was stimulated 20–30% by O₂ reduction, indicating the P_i‐regeneration limitation was removed during low‐temperature acclimation. In M. glomerata, in vitro Rubisco activity and gross CO₂ assimilation rate were equivalent below 25 °C, indicating that the capacity of the enzyme is a major rate limiting step during C₄ photosynthesis at cool temperatures.     

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.