Contents Summary 341 Introduction 342 What is C4 photosynthesis? 343 Why did C4 photosynthesis evolve? 347 Evolutionary lineages of C4 photosynthesis 348 Where did C4 photosynthesis evolve? 350 How did C4 photosynthesis evolve? 352 Molecular evolution of C4 photosynthesis 361 When did C4 photosynthesis evolve 362 The rise of C4 photosynthesis in relation to climate and CO2 363 Final thoughts: the future evolution of C4 photosynthesis 365 Acknowledgements 365 References 365 Summary C4 photosynthesis is a series of anatomical and biochemical modifications that concentrate CO2 around the carboxylating enzyme Rubisco, thereby increasing photosynthetic efficiency in conditions promoting high rates of photorespiration. The C4 pathway independently evolved over 45 times in 19 families of angiosperms, and thus represents one of the most convergent of evolutionary phenomena. Most origins of C4 photosynthesis occurred in the dicots, with at least 30 lineages. C4 photosynthesis first arose in grasses, probably during the Oligocene epoch (24–35 million yr ago). The earliest C4 dicots are likely members of the Chenopodiaceae dating back 15–21 million yr; however, most C4 dicot lineages are estimated to have appeared relatively recently, perhaps less than 5 million yr ago. C4 photosynthesis in the dicots originated in arid regions of low latitude, implicating combined effects of heat, drought and/or salinity as important conditions promoting C4 evolution. Low atmospheric CO2 is a significant contributing factor, because it is required for high rates of photorespiration. Consistently, the appearance of C4 plants in the evolutionary record coincides with periods of increasing global aridification and declining atmospheric CO2. Gene duplication followed by neo‐ and nonfunctionalization are the leading mechanisms for creating C4 genomes, with selection for carbon conservation traits under conditions promoting high photorespiration being the ultimate factor behind the origin of C4 photosynthesis.

We review the current understanding of the temperature responses of C3 and C4 photosynthesis across thermal ranges that do not harm the photosynthetic apparatus. In C3 species, photosynthesis is classically considered to be limited by the capacities of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco), ribulose bisphosphate (RuBP) regeneration or Pi regeneration. Using both theoretical and empirical evidence, we describe the temperature response of instantaneous net CO2 assimilation rate (A) in terms of these limitations, and evaluate possible limitations on A at elevated temperatures arising from heat-induced lability of Rubisco activase. In C3 plants, Rubisco capacity is the predominant limitation on A across a wide range of temperatures at low CO2 (<300 mbar), while at elevated CO2, the limitation shifts to Pi regeneration capacity at suboptimal temperatures, and either electron transport capacity or Rubisco activase capacity at supraoptimal temperatures. In C4 plants, Rubisco capacity limits A below 20°C in chilling-tolerant species, but the control over A at elevated temperature remains uncertain. Acclimation of C3 photosynthesis to suboptimal growth temperature is commonly associated with a disproportional enhancement of the Pi regeneration capacity. Above the thermal optimum, acclimation of A to increasing growth temperature is associated with increased electron transport capacity and/or greater heat stability of Rubisco activase. In many C4 species from warm habitats, acclimation to cooler growth conditions increases levels of Rubisco and C4 cycle enzymes which then enhance A below the thermal optimum. By contrast, few C4 species adapted to cooler habitats increase Rubisco content during acclimation to reduced growth temperature; as a result, A changes little at suboptimal temperatures. Global change is likely to cause a widespread shift in patterns of photosynthetic limitation in higher plants. Limitations in electron transport and Rubisco activase capacity should be more common in the warmer, high CO2 conditions expected by the end of the century.

C(4) photosynthesis is one of the most convergent evolutionary phenomena in the biological world, with at least 66 independent origins. Evidence from these lineages consistently indicates that the C(4) pathway is the end result of a series of evolutionary modifications to recover photorespired CO(2) in environments where RuBisCO oxygenation is high. Phylogenetically informed research indicates that the repositioning of mitochondria in the bundle sheath is one of the earliest steps in C(4) evolution, as it may establish a single-celled mechanism to scavenge photorespired CO(2) produced in the bundle sheath cells. Elaboration of this mechanism leads to the two-celled photorespiratory concentration mechanism known as C(2) photosynthesis (commonly observed in C(3)-C(4) intermediate species) and then to C(4) photosynthesis following the upregulation of a C(4) metabolic cycle.

The evolution of grasses using C4 photosynthesis and their sudden rise to ecological dominance 3 to 8 million years ago is among the most dramatic examples of biome assembly in the geological record. A growing body of work suggests that the patterns and drivers of C4 grassland expansion were considerably more complex than originally assumed. Previous research has benefited substantially from dialog between geologists and ecologists, but current research must now integrate fully with phylogenetics. A synthesis of grass evolutionary biology with grassland ecosystem science will further our knowledge of the evolution of traits that promote dominance in grassland systems and will provide a new context in which to evaluate the relative importance of C4 photosynthesis in transforming ecosystems across large regions of Earth.

The nature of photosynthetic acclimation to elevated CO2 is evaluated from the results of over 40 studies focusing on the effect of long-term CO2 enrichment on the short-term response of photosynthesis to intercellular CO2 (the A/Ci response). The effect of CO2 enrichment on the A/Ci response was dependent on growth conditions, with plants grown in small pots (< 5 L) or low nutrients usually exhibiting a reduction of A at a given Ci, while plants grown without nutrient deficiency in large pots or in the field tended to exhibit either little reduction or an enhancement of A at a given Ci following a doubling or tripling of atmospheric CO2 during growth. Using theoretical interpretations of A/Ci curves to assess acclimation, it was found that when pot size or nutrient deficiency was not a factor, changes in the shape of A/Ci curves which are indicative of a reallocation of resources within the photosynthetic apparatus typically were not observed. Long-term CO2 enrichment usually had little effect or increased the value of A at all Ci. However, a minority of species grown at elevated CO2 exhibited gas exchange responses indicative of a reduced amount of Rubisco and an enhanced capacity to metabolize photosynthetic products. This type of response was considered beneficial because it enhanced both photosynthetic capacity at high CO2 and reduced resource investment in excessive Rubisco capacity. The ratio of intercellular to ambient CO2 (the Ci/Ca ratio) was used to evaluate stomatal acclimation. Except under water and humidity stress, Ci/Ca exhibited no consistent change in a variety of C3 species, indicating no stomatal acclimation. Under drought or humidity stress, Ci/Ca declined in high-CO2 grown plants, indicating stomata will become more conservative during stress episodes in future high CO2 environments.

Using isotopic screens, phylogenetic assessments, and 45 years of physiological data, it is now possible to identify most of the evolutionary lineages expressing the C(4) photosynthetic pathway. Here, 62 recognizable lineages of C(4) photosynthesis are listed. Thirty-six lineages (60%) occur in the eudicots. Monocots account for 26 lineages, with a minimum of 18 lineages being present in the grass family and six in the sedge family. Species exhibiting the C(3)-C(4) intermediate type of photosynthesis correspond to 21 lineages. Of these, 9 are not immediately associated with any C(4) lineage, indicating that they did not share common C(3)-C(4) ancestors with C(4) species and are instead an independent line. The geographic centre of origin for 47 of the lineages could be estimated. These centres tend to cluster in areas corresponding to what are now arid to semi-arid regions of southwestern North America, south-central South America, central Asia, northeastern and southern Africa, and inland Australia. With 62 independent lineages, C(4) photosynthesis has to be considered one of the most convergent of the complex evolutionary phenomena on planet Earth, and is thus an outstanding system to study the mechanisms of evolutionary adaptation.

The effect of long-term (weeks to months) CO2 enhancement on (a) the gas-exchange characteristics, (b) the content and activation state of ribulose-1,5-bisphosphate carboxylase (rubisco), and (c) leaf nitrogen, chlorophyll, and dry weight per area were studied in five C3 species (Chenopodium album, Phaseolus vulgaris, Solanum tuberosum, Solanum melongena, and Brassica oleracea) grown at CO2 partial pressures of 300 or 900 to 1000 microbars. Long-term exposure to elevated CO2 affected the CO2 response of photosynthesis in one of three ways: (a) the initial slope of the CO2 response was unaffected, but the photosynthetic rate at high CO2 increased (S. tuberosum); (b) the initial slope decreased but the C02-saturated rate of photosynthesis was little affected (C. album, P. vulgaris); (c) both the initial slope and the C02-saturated rate of photosynthesis decreased (B. oleracea, S. melongena). In all five species, growth at high CO2 increased the extent to which photosynthesis was stimulated following a decrease in the partial pressure of 02 or an increase in measurement CO2 above 600 microbars. This stimulation indicates that a limitation on photosynthesis by the capacity to regenerate orthophosphate was reduced or absent after acclimation to high CO2. Leaf nitrogen per area either increased (S. tuberosum, S. melongena) or was little changed by CO2 enhancement. The content of rubisco was lower in only two of the five species, yet its activation state was 19% to 48% lower in all five species following longterm exposure to high CO2. These results indicate that during growth in C02-enriched air, leaf rubisco content remains in excess of that required to support the observed photosynthetic rates.During short-term (minutes to hours) exposure to elevated partial pressure of C02, the rate of light-saturated CO2 assimilation (A2) in many C3 plants is primarily limited by the capacity to regenerate Pi from phosphorylated photosynthetic intermediates (10, 17,22,23,26). A Pi-regeneration limitation of photosynthesis is characterized by a lack of sensitivity ofA to changes in ambient partial pressure of CO2 and/ or 02 (10,

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