Evolutionary transition from C3 to C4 photosynthesis and the route to C4 rice

Compared with C₃ plants, C₄ plants possess a mechanism to concentrate CO₂ around the ribulose-1,5-bisphosphate carboxylase/oxygenase in chloroplasts of bundle sheath cells so that the carboxylation reaction work at a much more efficient rate, thereby substantially eliminate the oxygenation reaction and the resulting photorespiration. It is observed that C₄ photosynthesis is more efficient than C₃ photosynthesis under conditions of low atmospheric CO₂, heat, drought and salinity, suggesting that these factors are the important drivers to promote C₄ evolution. Although C₄ evolution took over 66 times independently, it is hypothesized that it shared the following evolutionary trajectory: 1) gene duplication followed by neofunctionalization; 2) anatomical and ultrastructral changes of leaf architecture to improve the hydraulic systems; 3) establishment of two-celled photorespiratory pump; 4) addition of transport system; 5) co-option of the duplicated genes into C₄ pathway and adaptive changes of C₄ enzymes. Based on our current understanding on C₄ evolution, several strategies for engineering C₄ rice have been proposed to increase both photosynthetic efficiency and yield significantly in order to avoid international food crisis in the future, especially in the developing countries. Here we summarize the latest progresses on the studies of C₄ evolution and discuss the strategies to introduce two-celled C₄ pathway into rice.     

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.     

Evolution of the C4 photosynthetic pathway: events at the cellular and molecular levels

The biochemistry and leaf anatomy of plants using C₄ photosynthesis promote the concentration of atmospheric CO₂ in leaf tissue that leads to improvements in growth and yield of C₄ plants over C₃ species in hot, dry, high light, and/or saline environments. C₄ plants like maize and sugarcane are significant food, fodder, and bioenergy crops. The C₄ photosynthetic pathway is an excellent example of convergent evolution, having evolved in multiple independent lineages of land plants from ancestors employing C₃ photosynthesis. In addition to C₃ and C₄ species, some plant lineages contain closely related C₃–C₄ intermediate species that demonstrate leaf anatomical, biochemical, and physiological characteristics between those of C₃ plants and species using C₄ photosynthesis. These groups of plants have been extremely useful in dissecting the modifications to leaf anatomy and molecular biology, which led to the evolution of C₄ photosynthesis. It is now clear that great variation exists in C₄ leaf anatomy, and diverse molecular mechanisms underlie C₄ biochemistry and physiology. However, all these different paths have led to the same destination—the expression of a C₄ CO₂ concentrating mechanism. Further identification of C₄ leaf anatomical traits and molecular biological components, and understanding how they are controlled and assembled will not only allow for additional insights into evolutionary convergence, but also contribute to sustainable food and bioenergy production strategies.     

Evolution of C4 Photosynthesis in the Genus Flaveria: Establishment of a Photorespiratory CO2 Pump

C₄ photosynthesis is nature’s most efficient answer to the dual activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the resulting loss of CO₂ by photorespiration. Gly decarboxylase (GDC) is the key component of photorespiratory CO₂ release in plants and is active in all photosynthetic tissues of C₃ plants, but only in the bundle sheath cells of C₄ plants. The restriction of GDC to the bundle sheath is assumed to be an essential and early step in the evolution of C₄ photosynthesis, leading to a photorespiratory CO₂ concentrating mechanism. In this study, we analyzed how the P-protein of GDC (GLDP) became restricted to the bundle sheath during the transition from C₃ to C₄ photosynthesis in the genus Flaveria. We found that C₃ Flaveria species already contain a bundle sheath–expressed GLDP gene in addition to a ubiquitously expressed second gene, which became a pseudogene in C₄ Flaveria species. Analyses of C₃-C₄ intermediate Flaveria species revealed that the photorespiratory CO₂ pump was not established in one single step, but gradually. The knowledge gained by this study sheds light on the early steps in C₄ evolution.     

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.     

Variations in nitrogen use efficiency reflect the biochemical subtype while variations in water use efficiency reflect the evolutionary lineage of C4 grasses at inter‐glacial CO2

C₄ photosynthesis evolved multiple times in diverse lineages. Most physiological studies comparing C₄ plants were not conducted at the low atmospheric CO₂ prevailing during their evolution. Here, 24 C₄ grasses belonging to three biochemical subtypes [nicotinamide adenine dinucleotide malic enzyme (NAD‐ME), phosphoenolpyruvate carboxykinase (PCK) and nicotinamide adenine dinucleotide phosphate malic enzyme (NADP‐ME)] and six major evolutionary lineages were grown under ambient (400 μL L^?1) and inter‐glacial (280 μL L^?1) CO₂. We hypothesized that nitrogen‐related and water‐related physiological traits are associated with subtypes and lineages, respectively. Photosynthetic rate and stomatal conductance were constrained by the shared lineage, while variation in leaf mass per area (LMA), leaf N per area, plant dry mass and plant water use efficiency were influenced by the subtype. Subtype and lineage were equally important for explaining variations in photosynthetic nitrogen use efficiency (PNUE) and photosynthetic water use efficiency (PWUE). CO₂ treatment impacted most parameters. Overall, higher LMA and leaf N distinguished the Chloridoideae/NAD‐ME group, while NADP‐ME and PCK grasses were distinguished by higher PNUE regardless of lineage. Plants were characterized by high photosynthesis and PWUE when grown at ambient CO₂ and by high conductance at inter‐glacial CO₂. In conclusion, the evolutionary and biochemical diversity among C₄ grasses was aligned with discernible leaf physiology, but it remains unknown whether these traits represent ecophysiological adaptation.     

Role of Photorespiration and Cyclic Electron Transport in C<Subscript>4</Subscript> Photosynthesis Evolution in the C<Subscript>3</Subscript>–C<Subscript>4</Subscript> Intermediate Species <Emphasis Type="Italic">Sedobassia sedoides</Emphasis>

Plants from two Sedobassia sedoides (Pall.) Aschers populations (Makan and Valitovo) (Chenopodiaceae) with C₂ photosynthesis (precursor of C₄ photosynthesis in phylogenesis) and photorespiratory CO₂-concentrating mechanism were studied. Genetic polymorphism and isotope discrimination (δ¹³С) levels of the plants were determined under natural conditions, and their morpho-physiological parameters such as fresh and dry biomass of the above ground parts of plants, functioning of photosystem I (PSI) and photosystem II (PSII), intensity of net photosynthesis (A), transpiration (E), photorespiration and water use efficiency (WUE) of plants were calculated under control and salinine conditions (0 and 200 mM NaCl). Results of the population-genetic analysis showed that the Makan population is polymorphic (plastic) and the Valitovo population is monomorphic (narrowly specialized). There were no significant differences between the populations based on δ¹³С values or growth parameters, PSII, A, E and WUE under control conditions. Under saline conditions, dry biomass accumulation decreased in the Makan population by 15% and by more than 2- fold in the Valitovo population. Population differences were revealed in terms of photorespiration intensity and P700 oxidation kinetics under control and saline conditions. Under control conditions, Makan plants were characterized by a higher photorespiration intensity, which decreased by 2-fold under saline conditions to the photorespiration level of Valitovo plants. Cyclic electron transport activity was minimal in the control Makan plants, and it increased by almost 2-fold under saline conditions to the level of that in Valitovo plants under control and saline conditions. Under control conditions, photosynthesis in Makan plants can be specified as the proto-Kranz type (transitional type from C₃ to C₂) and that in Valitovo plants can be specified as the C₂ type (C₄ photosynthesis with photorespiratory CO₂-concentrating mechanism), based on their photorespiration level and cyclic electron transport activity. Under saline conditions, Makan plants exhibited features of C₂ photosynthesis. Intraspecific functional differences of photosynthesis were revealed in different populations of intermediate C₃–C₄ plant species S. sedoides which reflect the initial stages of formation of a photorespiratory CO₂-concentrating mechanism during C₄ photosynthesis evolution, accompanied by decrease in salt tolerance.     

C4 photosynthesis and water stress

Background

In contrast to C₃ photosynthesis, the response of C₄ photosynthesis to water stress has been less-well studied in spite of the significant contribution of C₄ plants to the global carbon budget and food security. The key feature of C₄ photosynthesis is the operation of a CO₂-concentrating mechanism in the leaves, which serves to saturate photosynthesis and suppress photorespiration in normal air. This article reviews the current state of understanding about the response of C₄ photosynthesis to water stress, including the interaction with elevated CO₂ concentration. Major gaps in our knowledge in this area are identified and further required research is suggested.

Scope

Evidence indicates that C₄ photosynthesis is highly sensitive to water stress. With declining leaf water status, CO₂ assimilation rate and stomatal conductance decrease rapidly and photosynthesis goes through three successive phases. The initial, mainly stomatal phase, may or may not be detected as a decline in assimilation rates depending on environmental conditions. This is because the CO₂-concentrating mechanism is capable of saturating C₄ photosynthesis under relatively low intercellular CO₂ concentrations. In addition, photorespired CO₂ is likely to be refixed before escaping the bundle sheath. This is followed by a mixed stomatal and non-stomatal phase and, finally, a mainly non-stomatal phase. The main non-stomatal factors include reduced activity of photosynthetic enzymes; inhibition of nitrate assimilation, induction of early senescence, and changes to the leaf anatomy and ultrastructure. Results from the literature about CO₂ enrichment indicate that when C₄ plants experience drought in their natural environment, elevated CO₂ concentration alleviates the effect of water stress on plant productivity indirectly via improved soil moisture and plant water status as a result of decreased stomatal conductance and reduced leaf transpiration.

Conclusions

It is suggested that there is a limited capacity for photorespiration or the Mehler reaction to act as significant alternative electron sinks under water stress in C₄ photosynthesis. This may explain why C₄ photosynthesis is equally or even more sensitive to water stress than its C₃ counterpart in spite of the greater capacity and water use efficiency of the C₄ photosynthetic pathway.Key words: C₃ and C₄ photosynthesis, stomatal and non-stomatal limitation, high CO₂, water stress     

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.     

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.     

C4 photosynthesis, atmospheric CO2, and climate

The objectives of this synthesis are (1) to review the factors that influence the ecological, geographical, and palaeoecological distributions of plants possessing C₄ photosynthesis and (2) to propose a hypothesis/model to explain both the distribution of C₄ plants with respect to temperature and CO₂ and why C₄ photosynthesis is relatively uncommon in dicotyledonous plants (hereafter dicots), especially in comparison with its widespread distribution in monocotyledonous species (hereafter monocots). Our goal is to stimulate discussion of the factors controlling distributions of C₄ plants today, historically, and under future elevated CO₂ environments. Understanding the distributions of C₃/C₄ plants impacts not only primary productivity, but also the distribution, evolution, and migration of both invertebrates and vertebrates that graze on these plants. Sixteen separate studies all indicate that the current distributions of C₄ monocots are tightly correlated with temperature: elevated temperatures during the growing season favor C₄ monocots. In contrast, the seven studies on C₄ dicot distributions suggest that a different environmental parameter, such as aridity (combination of temperature and evaporative potential), more closely describes their distributions. Differences in the temperature dependence of the quantum yield for CO₂ uptake (light-use efficiency) of C₃ and C₄ species relate well to observed plant distributions and light-use efficiency is the only mechanism that has been proposed to explain distributional differences in C₃/C₄ monocots. Modeling of C₃ and C₄ light-use efficiencies under different combinations of atmospheric CO₂ and temperature predicts that C₄-dominated ecosystems should not have expanded until atmospheric CO₂ concentrations reached the lower levels that are thought to have existed beginning near the end of the Miocene. At that time, palaeocarbonate and fossil data indicate a simultaneous, global expansion of C₄-dominated grasslands. The C₄ monocots generally have a higher quantum yield than C₄ dicots and it is proposed that leaf venation patterns play a role in increasing the light-use efficiency of most C₄ monocots. The reduced quantum yield of most C₄ dicots is consistent with their rarity, and it is suggested that C₄ dicots may not have been selected until CO₂ concentrations reached their lowest levels during glacial maxima in the Quaternary. Given the intrinsic light-use efficiency advantage of C₄ monocots, C₄ dicots may have been limited in their distributions to the warmest ecosystems, saline ecosystems, and/or to highly disturbed ecosystems. All C₄ plants have a significant advantage over C₃ plants under low atmospheric CO₂ conditions and are predicted to have expanded significantly on a global scale during full-glacial periods, especially in tropical regions. Bog and lake sediment cores as well as pedogenic carbonates support the hypothesis that C₄ ecosystems were more extensive during the last glacial maximum and then decreased in abundance following deglaciation as atmospheric CO₂ levels increased. Received: 12 February 1997 / Accepted: 20 June 1997     

Biochemical approaches to C4 photosynthesis evolution studies: the case of malic enzymes decarboxylases

C₄ photosynthesis enables the capture of atmospheric CO₂ and its concentration at the site of RuBisCO, thus counteracting the negative effects of low atmospheric levels of CO₂ and high atmospheric levels of O₂ (21 %) on photosynthesis. The evolution of this complex syndrome was a multistep process. It did not occur by simply recruiting pre-exiting components of the pathway from C₃ ancestors which were already optimized for C₄ function. Rather it involved modifications in the kinetics and regulatory properties of pre-existing isoforms of non-photosynthetic enzymes in C₃ plants. Thus, biochemical studies aimed at elucidating the functional adaptations of these enzymes are central to the development of an integrative view of the C₄ mechanism. In the present review, the most important biochemical approaches that we currently use to understand the evolution of the C₄ isoforms of malic enzyme are summarized. It is expected that this information will help in the rational design of the best decarboxylation processes to provide CO₂ for RuBisCO in engineering C₃ species to perform C₄ photosynthesis.     

Photosynthetic and photorespiratory characteristics of flaveria species

The genus Flaveria shows evidence of evolution in the mechanism of photosynthesis as its 21 species include C₃, C₃-C₄, C₄-like, and C₄ plants. In this study, several physiological and biochemical parameters of photosynthesis and photorespiration were measured in 18 Flaveria species representing all the photosynthetic types. The 10 species classified as C₃-C₄ intermediates showed an inverse continuum in level of photorespiration and development of the C₄ syndrome. This ranges from F. sonorensis with relatively high apparent photorespiration and lacking C₄ photosynthesis to F. Among the intermediates, the photosynthetic CO₂ compensation points at 30°C and 1150 micromoles quanta per square meter per second varied from 9 to 29 microbars. The values for the three C₄-like species varied from 3 to 6 microbars, similar to those measured for the C₄ species. The activities of the photorespiratory enzymes glycolate oxidase, hydroxypyruvate reductase, and serine hydroxymethyltransferase decreased progressively from C₃ to C₃-C₄ to C₄-like and C₄ species. On the other hand, most intermediates had higher levels of phosphenolpyruvate carboxylase and NADP-malic enzyme than C₃ species, but generally lower activities compared to C₄-like and C₄ species. The levels of these C₄ enzymes are correlated with the degree of C₄ photosynthesis, based on the initial products of photosynthesis. Another indication of development of the C₄ syndrome in C₃-C₄ Flaveria species was their intermediate chlorophyll a/b ratios. The chlorophyll a/b ratios of the various Flaveria species are highly correlated with the degree of C₄ photosynthesis suggesting that the photochemical machinery is progressively altered during evolution in order to meet the specific energy requirements for operating the C₄ pathway. In the progression from C₃ to C₄ species in Flaveria, the CO₂ compensation point decreased more rapidly than did the decrease in O₂ inhibition of photosynthesis or the increase in the degree of C₄ photosynthesis. These results suggest that the reduction in photorespiration during evolution occurred initially by refixation of photorespired CO₂ and prior to substantive reduction in O₂ inhibition and development of the C₄ syndrome. However, further reduction in O₂ inhibition in some intermediates and C₄-like species is considered primarily due to the development of the C₄ syndrome. Thus, the evolution of C₃-C₄ intermediate photosynthesis likely occurred in response to environmental conditions which limit the intercellular CO₂ concentration first via refixation of photorespired CO₂, followed by development of the C₄ syndrome.     

PHOTOSYNTHETIC CHARACTERISTICS OF C3-C4 INTERMEDIATE FLAVERIA FLORIDANA (ASTERACEAE) IN NATURAL HABITATS: EVIDENCE OF ADVANTAGES TO C3-C4 PHOTOSYNTHESIS AT HIGH LEAF TEMPERATURES

Measurements of leaf gas exchange were conducted in situ for the C₃-C₄ intermediate plant Flaveria floridana. Leaves exhibited measurable CO₂ assimilation at atmospheric CO₂ concentrations as low as 20 μmol/mol. This result demonstrates that the low CO₂ compensation points observed in past studies of greenhouse-grown C₃-C₄ intermediate plants also exist in plants growing in their natural habitat. Photosynthesis rates in F. floridana were near their maximum at intercellular CO₂ concentrations as low as 112 μmol/mol. The existence of near-maximum photosynthesis rates at such low intercellular CO₂ concentrations is interpreted as evidence for the existence of a CO₂-concentrating mechanism in F. floridana. Such a mechanism would also explain the observed lack of response in photosynthesis rates to reductions in stomatal conductance and intercellular CO₂ concentration as the leaf-to-air water vapor concentration gradient is increased. Photosynthetic rates were relatively high at leaf temperatures between 35 and 40 C, compared to most C₃ plants. At midday during May, when leaf temperatures were between 35 and 42 C, F. floridana leaves exhibited photosynthesis rates that were four times higher than a sympatric C₃ species (Eustoma exaltatum) of similar growth form and ecological habit. The high photosynthesis rates at high leaf temperatures in F. floridana were not due to higher leaf nitrogen contents, but rather to its reduced rate of photorespiration. These results confirm that C₃-C₄ intermediate photosynthesis can provide plants with an advantage at high leaf temperatures, compared to C₃ plants.     

Leaf development,gas exchange characteristics,and photorespiratory activity in maize seedlings

Five decades ago, a novel mode of CO₂ assimilation that was later described as C₄-photosynthesis was discovered on mature leaves of maize (Zea mays L.) plants. Here we show that 3- to 5-day-old developing maize leaves recapitulate the evolutionary advance from the ancient, inefficient C₃ mode of photosynthesis to the C₄ pathway, a mechanism for overcoming the wasteful process of photorespiration. Chlorophyll fluorescence measurements documented that photorespiration was high in 3-day-old juvenile primary leaves with non-specialized C₃-like leaf anatomy and low in 5-day-old organs with the typical “Kranz-anatomy” of C₄ leaves. Photosynthetic gas (CO₂)-exchange measurements on 5-day-old leaves revealed the characteristic features of C₄ photosynthesis, with a CO₂ compensation point close to zero and little inhibition of photosynthesis by the normal oxygen concentration in the air. This indicates a very low photorespiratory activity in contrast to control experiments conducted with mature C₃ sunflower (Helianthus annuus L.) leaves, which display a high rate of photorespiration.     

Crassulacean acid metabolism photosynthesis: `working the night shift'

Crassulacean acid metabolism (CAM) can be traced from Roman times through persons who noted a morning acid taste of some common house plants. From India in 1815, Benjamin-Heyne described a `daily acid taste cycle' with some succulent garden plants. Recent work has shown that the nocturnally formed acid is decarboxylated during the day to become the CO₂ for photosynthesis. Thus, CAM photosynthesis extends over a 24-hour day using several daily interlocking cycles. To understand CAM photosynthesis, several landmark discoveries were made at the following times: daily reciprocal acid and carbohydrate cycles were found during 1870 to 1887; their precise identification, as malic acid and starch, and accurate quantification occurred from 1940 to 1954; diffusive gas resistance methods were introduced in the early 1960s that led to understanding the powerful stomatal control of daily gas exchanges; C₄ photosynthesis in two different types of cells was discovered from 1965 to ∼1974 and the resultant information was used to elucidate the day and night portions of CAM photosynthesis in one cell; and exceptionally high internal green tissue CO₂ levels, 0.2 to 2.5%, upon the daytime decarboxylation of malic acid, were discovered in 1979. These discoveries then were combined with related information from C₃ and C₄ photosynthesis, carbon biochemistry, cellular anatomy, and ecological physiology. Therefore by ∼1980, CAM photosynthesis finally was rigorously outlined. In a nutshell, 24-hour CAM occurs by phosphoenol pyruvate (PEP) carboxylase fixing CO₂(HCO₃ ⁻) over the night to form malic acid that is stored in plant cell vacuoles. While stomata are tightly closed the following day, malic acid is decarboxylated releasing CO₂ for C₃ photosynthesis via ribulose bisphosphate carboxylase oxygenase (Rubisco). The CO₂ acceptor, PEP, is formed via glycolysis at night from starch or other stored carbohydrates and after decarboxylation the three carbons are restored each day. In mid to late afternoon the stomata can open and mostly C₃ photosynthesis occurs until darkness. CAM photo-synthesis can be both inducible and constitutive and is known in 33 families with an estimated 15 to 20 000 species. CAM plants express the most plastic and tenacious photosynthesis known in that they can switch photosynthesis pathways and they can live and conduct photosynthesis for years even in the virtual absence of external H₂O and CO₂, i.e., CAM tenaciously protects its photosynthesis from both H₂O and CO₂ stresses. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rubisco Evolution in C4 Eudicots: An Analysis of Amaranthaceae Sensu Lato

Background

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyses the key reaction in the photosynthetic assimilation of CO₂. In C₄ plants CO₂ is supplied to Rubisco by an auxiliary CO₂-concentrating pathway that helps to maximize the carboxylase activity of the enzyme while suppressing its oxygenase activity. As a consequence, C₄ Rubisco exhibits a higher maximum velocity but lower substrate specificity compared with the C₃ enzyme. Specific amino-acids in Rubisco are associated with C₄ photosynthesis in monocots, but it is not known whether selection has acted on Rubisco in a similar way in eudicots.

Methodology/Principal Findings

We investigated Rubisco evolution in Amaranthaceae sensu lato (including Chenopodiaceae), the third-largest family of C₄ plants, using phylogeny-based maximum likelihood and Bayesian methods to detect Darwinian selection on the chloroplast rbcL gene in a sample of 179 species. Two Rubisco residues, 281 and 309, were found to be under positive selection in C₄ Amaranthaceae with multiple parallel replacements of alanine by serine at position 281 and methionine by isoleucine at position 309. Remarkably, both amino-acids have been detected in other C₄ plant groups, such as C₄ monocots, illustrating a striking parallelism in molecular evolution.

Conclusions/Significance

Our findings illustrate how simple genetic changes can contribute to the evolution of photosynthesis and strengthen the hypothesis that parallel amino-acid replacements are associated with adaptive changes in Rubisco.     

Inorganic Carbon Diffusion between C(4) Mesophyll and Bundle Sheath Cells: Direct Bundle Sheath CO(2) Assimilation in Intact Leaves in the Presence of an Inhibitor of the C(4) Pathway

Photosynthesis rates of detached Panicum miliaceum leaves were measured, by either CO₂ assimilation or oxygen evolution, over a wide range of CO₂ concentrations before and after supplying the phosphoenolpyruvate (PEP) carboxylase inhibitor, 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate (DCDP). At a concentration of CO₂ near ambient, net photosynthesis was completely inhibited by DCDP, but could be largely restored by elevating the CO₂ concentration to about 0.8% (v/v) and above. Inhibition of isolated PEP carboxylase by DCDP was not competitive with respect to HCO₃⁻, indicating that the recovery was not due to reversal of enzyme inhibition. The kinetics of ¹⁴C-incorporation from ¹⁴CO₂ into early labeled products indicated that photosynthesis in DCDP-treated P. miliaceum leaves at 1% (v/v) CO₂ occurs predominantly by direct CO₂ fixation by ribulose 1,5-bisphosphate carboxylase. From the photosynthesis rates of DCDP-treated leaves at elevated CO₂ concentrations, permeability coefficients for CO₂ flux into bundle sheath cells were determined for a range of C₄ species. These values (6-21 micromoles per minute per milligram chlorophyll per millimolar, or 0.0016-0.0056 centimeter per second) were found to be about 100-fold lower than published values for mesophyll cells of C₃ plants. These results support the concept that a CO₂ permeability barrier exists to allow the development of high CO₂ concentrations in bundle sheath cells during C₄ photosynthesis.     

Upregulation of bundle sheath electron transport capacity under limiting light in C4 Setaria viridis

C₄ photosynthesis is a biochemical pathway that operates across mesophyll and bundle sheath (BS) cells to increase CO₂ concentration at the site of CO₂ fixation. C₄ plants benefit from high irradiance but their efficiency decreases under shade, causing a loss of productivity in crop canopies. We investigated shade acclimation responses of Setaria viridis, a model monocot of NADP-dependent malic enzyme subtype, focussing on cell-specific electron transport capacity. Plants grown under low light (LL) maintained CO₂ assimilation rates similar to high light plants but had an increased chlorophyll and light-harvesting-protein content, predominantly in BS cells. Photosystem II (PSII) protein abundance, oxygen-evolving activity and the PSII/PSI ratio were enhanced in LL BS cells, indicating a higher capacity for linear electron flow. Abundances of PSI, ATP synthase, Cytochrome b₆f and the chloroplast NAD(P)H dehydrogenase complex, which constitute the BS cyclic electron flow machinery, were also increased in LL plants. A decline in PEP carboxylase activity in mesophyll cells and a consequent shortage of reducing power in BS chloroplasts were associated with a more oxidised plastoquinone pool in LL plants and the formation of PSII – light-harvesting complex II supercomplexes with an increased oxygen evolution rate. Our results suggest that the supramolecular composition of PSII in BS cells is adjusted according to the redox state of the plastoquinone pool. This discovery contributes to the understanding of the acclimation of PSII activity in C₄ plants and will support the development of strategies for crop improvement, including the engineering of C₄ photosynthesis into C₃ plants.