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We crossed the C3 species Atriplex prostrata with the C4 species Atriplex rosea to produce F1 and F2 hybrids. All hybrids exhibited C3-like δ(13)C values, and had reduced rates of net CO2 assimilation compared with A. prostrata. The activities of the major C4 cycle enzymes PEP carboxylase, NAD-malic enzyme, and pyruvate-Pi dikinase in the hybrids were at most 36% of the C4 values. These results demonstrate the C4 metabolic cycle was disrupted in the hybrids. Photosynthetic CO2 compensation points (Г) of the hybrids were generally midway between the C3 and C4 values, and in most hybrids were accompanied by low, C3-like activities in one or more of the major C4 cycle enzymes. This supports the possibility that most hybrids use a photorespiratory glycine shuttle to concentrate CO2 into the bundle sheath cells. One hybrid exhibited a C4-like Г of 4 µmol mol(-1), indicating engagement of a C4 metabolic cycle. Consistently, this hybrid had elevated activities of all measured C4 cycle enzymes relative to the C3 parent; however, C3-like carbon isotope ratios indicate the low Г is mainly due to a photorespiratory glycine shuttle. The anatomy of the hybrids resembled that of C3-C4 intermediate species using a glycine shuttle to concentrate CO2 in the bundle sheath, and is further evidence that this physiology is the predominant, default condition of the F2 hybrids. Progeny of these hybrids should further segregate C3 and C4 traits and in doing so assist in the discovery of C4 genes using high-throughput methods of the genomics era., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Premise of the Study: Gisekiaceae are a monogeneric family of the core Caryophyllales distributed in arid regions of Africa and Asia. The only widespread species of the genus, Gisekia pharnaceoides, performs C4 photosynthesis based on CO2 compensation point measurements. This study investigates the C4 syndrome and its evolution in Gisekia. The infrageneric relationships, distribution and bioclimatic preferences of Gisekia are also investigated., Methods: Leaf gas exchange characteristics, activity of Rubisco and major C4 cycle enzymes, and ultrastructural characteristics of mesophyll and bundle sheath cells are studied for Gisekia pharnaceoides. δ(13)C values and leaf anatomy are analyzed for all species. A dated molecular phylogeny of 39 accessions representing all species of Gisekiaceae and 14 representatives of closely related core Caryophyllales families is generated using four cp markers and ITS. The precise current distribution and bioclimatic niche of Gisekia is assessed on the basis of 520 georeferenced specimen localities., Key Results: All traditionally recognized species of Gisekia are C4 plants with atriplicoid Kranz anatomy. Gisekia pharnaceoides uses the NAD-ME biochemical type. The molecular phylogeny demonstrated two East African clades nested within South African clades, demonstrating migration along the arid areas of eastern Africa during the Late Miocene/Pliocene Epochs. Most traditionally defined species are polyphyletic., Conclusions: Gisekia represents an isolated C4 lineage within core Caryophyllales dating back to the Miocene Epoch and probably spread along the African arid corridor from a South African center of origin. The seven currently recognized species should be treated as one polymorphic species or species complex, Gisekia pharnaceoides agg.

The evolution of C4 photosynthesis in many taxa involves the establishment of a two-celled photorespiratory CO2 pump, termed C2 photosynthesis. How C3 species evolved C2 metabolism is critical to understanding the initial phases of C4 plant evolution. To evaluate early events in C4 evolution, we compared leaf anatomy, ultrastructure, and gas-exchange responses of closely related C3 and C2 species of Flaveria, a model genus for C4 evolution. We hypothesized that Flaveria pringlei and Flaveria robusta, two C3 species that are most closely related to the C2 Flaveria species, would show rudimentary characteristics of C2 physiology. Compared with less-related C3 species, bundle sheath (BS) cells of F. pringlei and F. robusta had more mitochondria and chloroplasts, larger mitochondria, and proportionally more of these organelles located along the inner cell periphery. These patterns were similar, although generally less in magnitude, than those observed in the C2 species Flaveria angustifolia and Flaveria sonorensis. In F. pringlei and F. robusta, the CO2 compensation point of photosynthesis was slightly lower than in the less-related C3 species, indicating an increase in photosynthetic efficiency. This could occur because of enhanced refixation of photorespired CO2 by the centripetally positioned organelles in the BS cells. If the phylogenetic positions of F. pringlei and F. robusta reflect ancestral states, these results support a hypothesis that increased numbers of centripetally located organelles initiated a metabolic scavenging of photorespired CO2 within the BS. This could have facilitated the formation of a glycine shuttle between mesophyll and BS cells that characterizes C2 photosynthesis.

Photosynthetic carbon gain in plants using the C(3) photosynthetic pathway is substantially inhibited by photorespiration in warm environments, particularly in atmospheres with low CO(2) concentrations. Unlike C(4) plants, C(3) plants are thought to lack any mechanism to compensate for the loss of photosynthetic productivity caused by photorespiration. Here, for the first time, we demonstrate that the C(3) plants rice and wheat employ a specific mechanism to trap and reassimilate photorespired CO(2) . A continuous layer of chloroplasts covering the portion of the mesophyll cell periphery that is exposed to the intercellular air space creates a diffusion barrier for CO(2) exiting the cell. This facilitates the capture and reassimilation of photorespired CO(2) in the chloroplast stroma. In both species, 24-38% of photorespired and respired CO(2) were reassimilated within the cell, thereby boosting photosynthesis by 8-11% at ambient atmospheric CO(2) concentration and 17-33% at a CO(2) concentration of 200 µmol mol(-1) . Widespread use of this mechanism in tropical and subtropical C(3) plants could explain why the diversity of the world's C(3) flora, and dominance of terrestrial net primary productivity, was maintained during the Pleistocene, when atmospheric CO(2) concentrations fell below 200 µmol mol(-1) ., (© 2012 Blackwell Publishing Ltd.)

This study evaluates acclimation of photosynthesis and stomatal conductance in three evolutionary lineages of C(3), C(3)-C(4) intermediate, and C(4) species grown in the low CO(2) and hot conditions proposed to favo r the evolution of C(4) photosynthesis. Closely related C(3), C(3)-C(4), and C(4) species in the genera Flaveria, Heliotropium, and Alternanthera were grown near 380 and 180 μmol CO(2) mol(-1) air and day/night temperatures of 37/29°C. Growth CO(2) had no effect on photosynthetic capacity or nitrogen allocation to Rubisco and electron transport in any of the species. There was also no effect of growth CO(2) on photosynthetic and stomatal responses to intercellular CO(2) concentration. These results demonstrate little ability to acclimate to low CO(2) growth conditions in closely related C(3) and C(3)-C(4) species, indicating that, during past episodes of low CO(2), individual C(3) plants had little ability to adjust their photosynthetic physiology to compensate for carbon starvation. This deficiency could have favored selection for more efficient modes of carbon assimilation, such as C(3)-C(4) intermediacy. The C(3)-C(4) species had approximately 50% greater rates of net CO(2) assimilation than the C(3) species when measured at the growth conditions of 180 μmol mol(-1) and 37°C, demonstrating the superiority of the C(3)-C(4) pathway in low atmospheric CO(2) and hot climates of recent geological time.

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.

Next-generation sequencing plays a central role in the characterization and quantification of transcriptomes. Although numerous metrics are purported to quantify the quality of RNA, there have been no large-scale empirical evaluations of the major determinants of sequencing success. We used a combination of existing and newly developed methods to isolate total RNA from 1115 samples from 695 plant species in 324 families, which represents >900 million years of phylogenetic diversity from green algae through flowering plants, including many plants of economic importance. We then sequenced 629 of these samples on Illumina GAIIx and HiSeq platforms and performed a large comparative analysis to identify predictors of RNA quality and the diversity of putative genes (scaffolds) expressed within samples. Tissue types (e.g., leaf vs. flower) varied in RNA quality, sequencing depth and the number of scaffolds. Tissue age also influenced RNA quality but not the number of scaffolds ≥ 1000 bp. Overall, 36% of the variation in the number of scaffolds was explained by metrics of RNA integrity (RIN score), RNA purity (OD 260/230), sequencing platform (GAIIx vs HiSeq) and the amount of total RNA used for sequencing. However, our results show that the most commonly used measures of RNA quality (e.g., RIN) are weak predictors of the number of scaffolds because Illumina sequencing is robust to variation in RNA quality. These results provide novel insight into the methods that are most important in isolating high quality RNA for sequencing and assembling plant transcriptomes. The methods and recommendations provided here could increase the efficiency and decrease the cost of RNA sequencing for individual labs and genome centers.

Photosynthetic pathway characteristics were studied in nine species of Heliotropium (sensu lato, including Euploca), using assessments of leaf anatomy and ultrastructure, activities of PEP carboxylase and C₄ acid decarboxylases, and immunolocalization of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and the P-subunit of glycine decarboxylase (GDC). Heliotropium europaeum, Heliotropium calcicola and Heliotropium tenellum are C₃ plants, while Heliotropium texanum and Heliotropium polyphyllum are C₄ species. Heliotropium procumbens and Heliotropium karwinskyi are functionally C₃, but exhibit 'proto-Kranz' anatomy where bundle sheath (BS) cells are enlarged and mitochondria primarily occur along the centripetal (inner) wall of the BS cells; GDC is present throughout the leaf. Heliotropium convolvulaceum and Heliotropium greggii are C₃--C₄ intermediates, with Kranz-like enlargement of the BS cells, localization of mitochondria along the inner BS wall and a loss of GDC in the mesophyll (M) tissue. These C₃--C₄ species of Heliotropium probably shuttle photorespiratory glycine from the M to the BS tissue for decarboxylation. Heliotropium represents an important new model for studying C₄ evolution. Where existing models such as Flaveria emphasize diversification of C₃--C₄ intermediates, Heliotropium has numerous C₃ species expressing proto-Kranz traits that could represent a critical initial phase in the evolutionary origin of C₄ photosynthesis., (© 2011 Blackwell Publishing Ltd.)

Plants using the C(4) pathway of carbon metabolism are marked by greater photosynthetic water and nitrogen-use efficiencies (PWUE and PNUE, respectively) than C(3) species, but it is unclear to what extent this is the case in C(3) -C(4) intermediate species. In this study, we examined the PWUE and PNUE of 14 species of Flaveria Juss. (Asteraceae), including two C(3) , three C(4) and nine C(3) -C(4) species, the latter containing a gradient of C(4) -cycle activities (as determined by initial fixation of (14) C into C-4 acids). We found that PWUE, PNUE, leaf ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) content and intercellular CO(2) concentration in air (C(i) ) do not change gradually with C(4) -cycle activity. These traits were not significantly different between C(3) species and C(3) -C(4) species with less than 50% C(4) -cycle activity. C(4) -like intermediates with greater than 65% C(4) -cycle activity were not significantly different from plants with fully expressed C(4) photosynthesis. These results indicate that a gradual increase in C(4) -cycle activity has not resulted in a gradual change in PWUE, PNUE, intercellular CO(2) concentration and leaf Rubisco content towards C(4) levels in the intermediate species. Rather, these traits arose in a stepwise manner during the evolutionary transition to the C(4) -like intermediates, which are contained in two different clades within Flaveria., (© 2011 Blackwell Publishing Ltd.)

Ever since the discovery of C(4) photosynthesis in the mid-1960s, plant biologists have envisaged the introduction of the C(4) photosynthetic pathway into C(3) crops such as rice and soybeans. Recent advances in genomics capabilities, and new evolutionary and developmental studies indicate that C(4) engineering will be feasible in the next few decades. Furthermore, better understanding of the function of C(4) photosynthesis provides new ways to improve existing C(4) crops and bioenergy species, for example by creating varieties with ultra-high water and nitrogen use efficiencies. In the case of C(4) engineering, the main enzymes of the C(4) metabolic cycle have already been engineered into various C(3) plants. In contrast, knowledge of the genes controlling Kranz anatomy lags far behind. Combining traditional genetics, high-throughput sequencing technologies, systems biology, bioinformatics, and the use of the new C(4) model species Setaria viridis, the discovery of the key genes controlling the expression of C(4) photosynthesis can be dramatically accelerated. Sustained investment in the research areas directly related to C(4) engineering has the potential for substantial return in the decades to come, primarily by increasing crop production at a time when global food supplies are predicted to fall below world demand.

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.

This study investigated whether Euphorbia subgenus Chamaesyce subsection Acutae contains C(3)-C(4) intermediate species utilizing C(2) photosynthesis, the process where photorespired CO(2) is concentrated into bundle sheath cells. Euphorbia species in subgenus Chamaesyce are generally C(4), but three species in subsection Acutae (E. acuta, E. angusta, and E. johnstonii) have C(3) isotopic ratios. Phylogenetically, subsection Acutae branches between basal C(3) clades within Euphorbia and the C(4) clade in subgenus Chamaesyce. Euphorbia angusta is C(3), as indicated by a photosynthetic CO(2) compensation point (Г) of 69 μmol mol(-1) at 30 °C, a lack of Kranz anatomy, and the occurrence of glycine decarboxylase in mesophyll tissues. Euphorbia acuta utilizes C(2) photosynthesis, as indicated by a Г of 33 μmol mol(-1) at 30 °C, Kranz-like anatomy with mitochondria restricted to the centripetal (inner) wall of the bundle sheath cells, and localization of glycine decarboxlyase to bundle sheath mitochondria. Low activities of PEP carboxylase, NADP malic enzyme, and NAD malic enzyme demonstrated no C(4) cycle activity occurs in E. acuta thereby classifying it as a Type I C(3)-C(4) intermediate. Kranz-like anatomy in E. johnstonii indicates it also utilizes C(2) photosynthesis. Given the phylogenetically intermediate position of E. acuta and E. johnstonii, these results support the hypothesis that C(2) photosynthesis is an evolutionary intermediate condition between C(3) and C(4) photosynthesis.

C(4) photosynthesis is a plant adaptation to high levels of photorespiration. Physiological models predict that atmospheric CO(2) concentration selected for C(4) grasses only after it dropped below a critical threshold during the Oligocene (∼30 Ma), a hypothesis supported by phylogenetic and molecular dating analyses. However the same models predict that CO(2) should have reached much lower levels before selecting for C(4) eudicots, making C(4) eudicots younger than C(4) grasses. In this study, different phylogenetic datasets were combined in order to conduct the first comparative analysis of the age of C(4) origins in eudicots. Our results suggested that all lineages of C(4) eudicots arose during the last 30 million years, with the earliest before 22 Ma in Chenopodiaceae and Aizoaceae, and the latest probably after 2 Ma in Flaveria. C(4) eudicots are thus not globally younger than C(4) monocots. All lineages of C(4) plants evolved in a similar low CO(2) atmosphere that predominated during the last 30 million years. Independent C(4) origins were probably driven by different combinations of specific factors, including local ecological characteristics such as habitat openness, aridity, and salinity, as well as the speciation and dispersal history of each clade. Neither the lower number of C(4) species nor the frequency of C(3)-C(4) intermediates in eudicots can be attributed to a more recent origin, but probably result from variation in diversification and evolutionary rates among the different groups that evolved the C(4) pathway.

C(4) photosynthesis is a series of biochemical and structural modifications to C(3) photosynthesis that has evolved numerous times in flowering plants, despite requiring modification of up to hundreds of genes. To study the origin of C(4) photosynthesis, we reconstructed and dated the phylogeny of Molluginaceae, and identified C(4) taxa in the family. Two C(4) species, and three clades with traits intermediate between C(3) and C(4) plants were observed in Molluginaceae. C(3)-C(4) intermediacy evolved at least twice, and in at least one lineage was maintained for several million years. Analyses of the genes for phosphoenolpyruvate carboxylase, a key C(4) enzyme, indicate two independent origins of fully developed C(4) photosynthesis in the past 10 million years, both within what was previously classified as a single species, Mollugo cerviana. The propensity of Molluginaceae to evolve C(3)-C(4) and C(4) photosynthesis is likely due to several traits that acted as developmental enablers. Enlarged bundle sheath cells predisposed some lineages for the evolution of C(3)-C(4) intermediacy and the C(4) biochemistry emerged via co-option of photorespiratory recycling in C(3)-C(4) intermediates. These evolutionarily stable transitional stages likely increased the evolvability of C(4) photosynthesis under selection environments brought on by climate and atmospheric change in recent geological time., (© 2010 The Author(s). Evolution© 2010 The Society for the Study of Evolution.)

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.

One mechanism to enhance global food stocks radically is to introduce C4 photosynthesis into C3 crops from warm climates, notably rice. To accomplish this, an understanding of leaf structure and function is essential. The chlorenchyma structure of rice and related warm-climate C3 grasses is distinct from that of cool temperate C3 grasses. In temperate C3 grasses, vacuoles occupy the majority of the cell, while chloroplasts, peroxisomes and mitochondria are pressed against the cell periphery. In rice, 66% of protoplast volume is occupied by chloroplasts, and chloroplasts/stromules cover >95% of the cell periphery. Mitochondria and peroxisomes occur in the cell interior and are intimately associated with chloroplasts/stromules. We hypothesize that the chlorenchyma architecture of rice enhances diffusive CO(2) conductance and maximizes scavenging of photorespired CO2. The extensive chloroplast/stromule sheath forces photorespired CO(2) to exit cells via the stroma, where it can be refixed by Rubisco. Deep cell lobing and small cell size, coupled with chloroplast sheaths, creates high surface area exposure of stroma to intercellular spaces, thereby enhancing mesophyll transfer conductance. In support of this, rice exhibits higher mesophyll transfer conductance, greater stromal CO2 content, lower CO2 compensation points at warm temperature and less oxygen sensitivity of photosynthesis than cool temperate grasses. Rice vein length per leaf, mesophyll thickness and intercellular space volume are intermediate between those of most C3 and C4 grasses, indicating that the introduction of Kranz anatomy into rice may not require radical changes in leaf anatomy; however, deep lobing of chlorenchyma cells may constrain efforts to engineer C4 photosynthesis into rice.

Higher water use efficiency (WUE) in C(4) plants may allow for greater xylem safety because transpiration rates are reduced. To evaluate this hypothesis, stem hydraulics and anatomy were compared in 16 C(3), C(3)-C(4) intermediate, C(4)-like and C(4) species in the genus Flaveria. The C(3) species had the highest leaf-specific conductivity (K(L)) compared with intermediate and C(4) species, with the perennial C(4) and C(4)-like species having the lowest K(L) values. Xylem-specific conductivity (K(S)) was generally highest in the C(3) species and lower in intermediate and C(4) species. Xylem vessels were shorter, narrower and more frequent in C(3)-C(4) intermediate, C(4)-like and C(4) species compared with C(3) species. WUE values were approximately double in the C(4)-like and C(4) species relative to the C(3)-C(4) and C(3) species. C(4)-like photosynthesis arose independently at least twice in Flaveria, and the trends in WUE and K(L) were consistent in both lineages. These correlated changes in WUE and K(L) indicate WUE increase promoted K(L) decline during C(4) evolution; however, any involvement of WUE comes late in the evolutionary sequence. C(3)-C(4) species exhibited reduced K(L) but little change in WUE compared to C(3) species, indicating that some reduction in hydraulic efficiency preceded increases in WUE.

We investigated the thermal acclimation of photosynthesis and respiration in black spruce seedlings [Picea mariana (Mill.) B.S.P.] grown at 22/14 degrees C [low temperature (LT)] or 30/22 degrees C [high temperature (HT)] day/night temperatures. Net CO(2) assimilation rates (A(net)) were greater in LT than in HT seedlings below 30 degrees C, but were greater in HT seedlings above 30 degrees C. Dark and day respiration rates were similar between treatments at the respective growth temperatures. When respiration was factored out of the photosynthesis response to temperature, the resulting gross CO(2) assimilation rates (A(gross)) was lower in HT than in LT seedlings below 30 degrees C, but was similar above 30 degrees C. The reduced A(gross) of HT seedlings was associated with lower needle nitrogen content, lower ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) maximum carboxylation rates (V(cmax)) and lower maximum electron transport rates (J(max)). Growth treatment did not affect V(cmax) : J(max). Modelling of the CO(2) response of photosynthesis indicated that LT seedlings at 40 degrees C might have been limited by heat lability of Rubisco activase, but that in HT seedlings, Rubisco capacity was limiting. In sum, thermal acclimation of A(net) was largely caused by reduced respiration and lower nitrogen investments in needles from HT seedlings. At 40 degrees C, photosynthesis in LT seedlings might be limited by Rubisco activase capacity, while in HT seedlings, acclimation removed this limitation.

The reasons for the decline in net CO2 assimilation (A) above its thermal optimum are controversial. We tested the hypothesis that increasing the ratio of Rubisco activase to Rubisco catalytic site concentration would increase the activation state of Rubisco at high temperatures. We measured photosynthetic gas exchange, in vivo electron transport (J) and the activation state of Rubisco between 15 and 45 degrees C, at 38 and 76 Pa ambient CO2, in wild-type (WT) and anti-rbcS tobacco. The Rubisco content of the anti-rbcS lines was 30% (S7-1) or 6% (S7-2) of WT, but activase levels were the same in the three genotypes. Anti-rbcS plants had lower A than WT at all temperatures, but had a similar thermal optimum for photosynthesis as WT at both CO2 levels. In WT plants, Rubisco was fully activated at 32 degrees C, but the activation state declined to 64% at 42 degrees C. By contrast, the activation state of Rubisco was above 90% in the S7-1 line, between 15 and 42 degrees C. Both A and J declined about 20% from T(opt) to the highest measurement temperatures in WT and the S7-1 line, but this was fully reversed after a 20 min recovery at 35 degrees C. At 76 Pa CO2, predicted rates of RuBP regeneration-limited photosynthesis corresponded with measured A in WT tobacco at all temperatures, and in S7-1 tobacco above 40 degrees C. Our observations are consistent with the hypothesis that the high temperature decline in A in the WT is because of an RuBP regeneration limitation, rather than the capacity of Rubisco activase to maintain high Rubisco activation state.

Global warming and the rise in atmospheric CO(2) will increase the operating temperature of leaves in coming decades, often well above the thermal optimum for photosynthesis. Presently, there is controversy over the limiting processes controlling photosynthesis at elevated temperature. Leading models propose that the reduction in photosynthesis at elevated temperature is a function of either declining capacity of electron transport to regenerate RuBP, or reductions in the capacity of Rubisco activase to maintain Rubisco in an active configuration. Identifying which of these processes is the principal limitation at elevated temperature is complicated because each may be regulated in response to a limitation in the other. Biochemical and gas exchange assessments can disentangle these photosynthetic limitations; however, comprehensive assessments are often difficult and, for many species, virtually impossible. It is proposed that measurement of the initial slope of the CO(2) response of photosynthesis (the A/C(i) response) can be a useful means to screen for Rubisco activase limitations. This is because a reduction in the Rubisco activation state should be most apparent at low CO(2) when Rubisco capacity is generally limiting. In sweet potato, spinach, and tobacco, the initial slope of the A/C(i) response shows no evidence of activase limitations at high temperature, as the slope can be accurately modelled using the kinetic parameters of fully activated Rubisco. In black spruce (Picea mariana), a reduction in the initial slope above 30 degrees C cannot be explained by the known kinetics of fully activated Rubisco, indicating that activase may be limiting at high temperatures. Because black spruce is the dominant species in the boreal forest of North America, Rubisco activase may be an unusually important factor determining the response of the boreal biome to climate change.

Increased cell size, increased leaf succulence, reduced intercellular air space (IAS), and reduced surface of mesophyll exposed to IAS (L(mes)/area) are traits associated with the Crassulacean acid metabolism (CAM) photosynthetic pathway. An examination was carried out to determine whether these anatomical and structural traits are related to the degree of CAM function in eight CAM species, as measured by CO(2) assimilation during the CAM and C(3) phases. Increased cell size and leaf succulence were not closely related to the degree of CAM function, indicating that succulence beyond a certain threshold does not enhance CAM function. Reduced IAS and L(mes)/area were positively related to CAM function, and negatively related to C(3) function. These results indicate that reduced IAS and L(mes)/area are beneficial for CAM function through the reduction of CO(2) efflux and the improvement of carbon economy. However, reduced IAS and L(mes)/area limit C(3) photosynthesis, potentially mediating a bimodal distribution of weak and strong CAM species with high and low IAS and L(mes)/area values, respectively.

The Portulacaceae is one of the few terrestrial plant families known to have both C(4) and Crassulacean acid metabolism (CAM) species. There may be multiple origins of the evolution of CAM within the Portulacaceae but the only clear evidence of C(4) photosynthesis is found in members of the genus Portulaca. In the Portulaca, CAM succulent tissue is overlaid with the C(4) tissue in a unique fashion where both pathways are operating simultaneously. Earlier reports have shown that the clade containing the genera Anacampseros and Grahamia may also contain C(4) photosynthetic species similar to the Portulaca, which would indicate multiple origins of C(4) photosynthesis within the family. The aim of the present study was to ascertain the true photosynthetic nature of these genera. An initial survey of the carbon isotope composition of the Anacampseros ranged from -12.6 per thousand to -24.0 per thousand, indicating very little CAM activity in some species, with other values close to the C(4) range. Anacampseros (=Grahamia) australiana which had been previously identified as a C(4) species had a carbon isotope composition value of -24.0 per thousand, which is more indicative of a C(3) species with a slight contribution of CAM activity. Other Anacampseros species with C(4)-like values have been shown to be CAM plants. The initial isotope analysis of the Grahamia species gave values in the range of -27.1 per thousand to -23.6 per thousand, placing the Grahamia species well towards the C(3) photosynthetic range. Further physiological studies indicated increased night-time CO(2) uptake with imposition of water stress, associated with a large diurnal acid fluctuation and a marked increased phosphoenolpyruvate carboxylase activity. This showed that the Grahamia species are actually facultative CAM plants despite their C(3)-like carbon isotope values. The results indicate that the Grahamia and Anacampseros species do not utilize the C(4) photosynthetic pathway. This is the first to identify that the Grahamia species are facultative CAM plants where CAM can be induced by water stress. This work supports earlier physiological work that indicates that this clade containing Anacampseros and Grahamia species comprises predominantly facultative CAM plants. This report suggests there may be only one clade which contains C(4) photosynthetic members with CAM-like characteristics.

C(4) photosynthesis evolved multiple times in the Amaranthaceae s.s., but the C(4) evolutionary lineages are unclear because the photosynthetic pathway is unknown for most species of the family. To clarify the distribution of C(4) photosynthesis in the Amaranthaceae, we determined carbon isotope ratios of 607 species and mapped these onto a phylogeny determined from matK/trnK sequences. Approximately 28% of the Amaranthaceae species use the C(4) pathway. C(4) species occur in 10 genera-Aerva, Amaranthus, Blutaparon, Alternanthera, Froelichia, Lithophila, Guilleminea, Gomphrena, Gossypianthus, and Tidestromia. Aerva, Alternanthera, and Gomphrena contain both C(3) and C(4) species. In Aerva, 25% of the sampled species are C(4). In Alternanthera, 19.5% are C(4), while 89% of the Gomphrena species are C(4). Integration of isotope and matK/trnK data indicated C(4) photosynthesis evolved five times in the Amaranthaceae, specifically in Aerva, Alternanthera, Amaranthus, Tidestromia, and a lineage containing Froelichia, Blutaparon, Guilleminea, Gomphrena pro parte, and Lithophila. Aerva and Gomphrena are both polyphyletic with C(3) and C(4) species belonging to distinct clades. Alternanthera appears to be monophyletic with C(4) photosynthesis originating in a terminal sublineage of procumbent herbs. Alpine C(4) species were also identified in Alternanthera, Amaranthus, and Gomphrena, including one species (Gomphrena meyeniana) from 4600 m a.s.l.

We demonstrate for the first time the presence of species exhibiting C3-C4 intermediacy in Heliotropium (sensu lato), a genus with over 100 C3 and 150 C4 species. CO2 compensation points (Gamma) and photosynthetic water-use efficiencies (WUEs) were intermediate between C3 and C4 values in three species of Heliotropium: Heliotropium convolvulaceum (Gamma = 20 micromol CO2 mol(-1) air), Heliotropium racemosum (Gamma = 22 micromol mol(-1)) and Heliotropium greggii (Gamma = 17 micromol mol(-1)). Heliotropium procumbens may also be a weak C3-C4 intermediate based on a slight reduction in Gamma (48.5 micromol CO2 mol(-1)) compared to C3Heliotropium species (52-60 micromol mol(-1)). The intermediate species H. convolvulaceum, H. greggii and H. racemosum exhibited over 50% enhancement of net CO2 assimilation rates at low CO2 levels (200-300 micromol mol(-1)); however, no significant differences in stomatal conductance were observed between the C3 and C3-C4 species. We also assessed the response of Gamma to variation in O2 concentration for these species. Heliotropium convolvulaceum, H. greggii and H. racemosum exhibited similar responses of Gamma to O2 with response slopes that were intermediate between the responses of C3 and C4 species below 210 mmol O2 mol(-1) air. The presence of multiple species displaying C3-C4 intermediate traits indicates that Heliotropium could be a valuable new model for studying the evolutionary transition from C3 to C4 photosynthesis.

The responses of chlorophyll fluorescence, gas exchange rate and Rubisco activation state to temperature were examined in transgenic rice plants with 130 and 35% of the wild-type (WT) Rubisco content by transformation with rbcS cDNA in sense and antisense orientations, respectively. Although the optimal temperatures of PSII quantum efficiency and CO(2) assimilation were found to be between 25 and 32 degrees C, the maximal activation state of Rubisco was found to be between 16 and 20 degrees C in all genotypes. The Rubisco flux control coefficient was also the highest between 16 and 20 degrees C in the WT and antisense lines [>0.88 at an intercellular CO(2) pressure (Ci) of 28 Pa]. Gross photosynthesis at Ci = 28 Pa per Rubisco content in the WT between 12 and 20 degrees C was close to that of the antisense lines where high Rubisco control is present. Thus, Rubisco activity most strongly limited photosynthesis at cool temperatures. These results indicated that a selective enhancement of Rubisco content can enhance photosynthesis at cool temperatures, but in the sense line with enhanced Rubisco content Pi regeneration limitation occurred. Above 20 degrees C, the Rubisco flux control coefficient declined. This decline was associated with a decline in Rubisco activation. The activation state of Rubisco measured at each temperature decreased with increasing Rubisco content, and the slope of activation to Rubisco content was independent of temperature. We discuss the possibility that the decline in Rubisco activation at intermediate and high temperatures is part of a regulated response to a limitation in other photosynthetic processes.

C(4) photosynthesis involves alterations to leaf development, cell biology and biochemistry. Different lineages of C(4) plants use varying mechanisms to generate the C(4) pathway. Although the biochemistry of C(4) photosynthesis was described around 20 years ago, the phylogenetic distance between Arabidopsis and the traditional C(4) models has not facilitated the transfer of knowledge from Arabidopsis research to understanding C(4) systems. We show that Cleome, a genus closely related to Arabidopsis, contains species spanning a developmental progression from C(3) to C(4) photosynthesis. The majority of species we assessed are C(3) plants but have increased venation in leaves. Three C(3) species have both increased venation and enlarged bundle sheath cells, and there is also a tendency to accumulate proteins and transcripts needed for C(4) photosynthesis. Cleome gynandra shows all the characteristics needed for efficient C(4) photosynthesis, including alterations to leaf biochemistry, cell biology and development, and belongs to the NAD-dependent malic enzyme subtype. Combined with its phylogenetic proximity to Arabidopsis, the developmental progression from C(3) to C(4) photosynthesis within the genus provides a potentially excellent new model to increase our understanding of C(4) photosynthesis, and provide insights into its evolution.

We review the current understanding of the temperature responses of C(3) and C(4) photosynthesis across thermal ranges that do not harm the photosynthetic apparatus. In C(3) species, photosynthesis is classically considered to be limited by the capacities of ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco), ribulose bisphosphate (RuBP) regeneration or P(i) regeneration. Using both theoretical and empirical evidence, we describe the temperature response of instantaneous net CO(2) 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 C(3) plants, Rubisco capacity is the predominant limitation on A across a wide range of temperatures at low CO(2) (<300 microbar), while at elevated CO(2), the limitation shifts to P(i) regeneration capacity at suboptimal temperatures, and either electron transport capacity or Rubisco activase capacity at supraoptimal temperatures. In C(4) plants, Rubisco capacity limits A below 20 degrees C in chilling-tolerant species, but the control over A at elevated temperature remains uncertain. Acclimation of C(3) photosynthesis to suboptimal growth temperature is commonly associated with a disproportional enhancement of the P(i) 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 C(4) species from warm habitats, acclimation to cooler growth conditions increases levels of Rubisco and C(4) cycle enzymes which then enhance A below the thermal optimum. By contrast, few C(4) 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 CO(2) conditions expected by the end of the century.

C(4) photosynthesis and Kranz anatomy occur in 16 eudicot families, a striking example of convergent evolution. Biochemical subtyping for 13 previously undiagnosed C(4) eudicot species indicated that 10 were NADP-malic enzyme (ME) and three were NAD-ME. A total of 33 C(4) species, encompassing four Kranz anatomical types (atriplicoid, kochioid, salsoloid, and suaedioid), and 21 closely related C(3) species were included in a quantitative anatomical study in which we found that, unlike similar studies in grasses and sedges, anatomical type had no predictive value for the biochemical subtype. In a multivariate canonical discriminant analysis, C(4) species were distinguished from C(3) species by the mesophyll to bundle sheath ratio and exposure of the bundle sheath surface to intercellular space. Discrimination between NADP-ME and NAD-ME was not significant, although in a Mantel test grouping by biochemical subtype was significant, while grouping by family was not. This comprehensive survey of C(4) anatomy and biochemistry unequivocally demonstrated that atriplicoid anatomy and NADP-ME biochemistry predominate in many evolutionary lineages. In addition to a main decarboxylating enzyme, high activity of a second decarboxylating enzyme was often observed. Notably, PEP-carboxykinase activity was significant in a number of species, demonstrating that this enzyme could also serve as a secondary pathway for C(4) metabolism in eudicots.

Flaveria (Asteraceae) is one of the few genera known to contain both C 3 and C 4 species, in addition to numerous biochemically-intermediate species. C 3 -C 4 and C 4 -like intermediate photosynthesis have arisen more than once in different phylogenetic clades of Flaveria. Here, we characterise for the first time the photosynthetic pathway of the recently described species Flaveria kochiana B.L. Turner. We examined leaf anatomy, activity and localisation of key photosynthetic enzymes, and gas exchange characteristics and compared these trait values with those from related C 4 and C 4 -like Flaveria species. F. kochiana has Kranz anatomy that is typical of other C 4 Flaveria species. As in the other C 4 lineages within the Flaveria genus, the primary decarboxylating enzyme is NADP-malic enzyme. Immunolocalisation of the major C 4 cycle enzymes, PEP carboxylase and pyruvate, orthophosphate dikinase, were restricted to the mesophyll, while Rubisco was largely localised to the bundle sheath. Gas exchange analysis demonstrated that F. kochiana operates a fully functional C 4 pathway with little sensitivity to ambient oxygen levels. The CO 2 compensation point (2.2 µbar) was typical for C 4 species, and the O 2 -response of the CO 2 compensation point was the same as the C 4 species F. trinervia. Notably, F. vaginata (B.L. Robinson & Greenman), a putative C 4 -like species that is the nearest relative of F. kochiana, had an identical response of the CO 2 compensation point to O 2 . Furthermore, F. vaginata, exhibited a carbon isotope ratio (-15.4‰) similar to C 4 species including F. australasica Hooker, F. trinervia Spreng. C. Mohr and the newly characterised F. kochiana. F. vaginata could be considered a C 4 species, but additional studies are necessary to confirm this hypothesis. In addition, our results show that F. kochiana uses an efficient C 4 cycle, with the highest initial slope of the A/C i curve of any C 4 Flaveria species.

C4 photosynthesis is a complex specialization that enhances carbon gain in hot, often arid habitats where photorespiration rates can be high. Certain features unique to C4 photosynthesis may reduce the potential for phenotypic plasticity and photosynthetic acclimation to environmental change relative to what is possible with C3 photosynthesis. During acclimation, the structural and physiological integrity of the mesophyll-bundle sheath (M-BS) complex has to be maintained if C4 photosynthesis is to function efficiently in the new environment. Disruption of the M-BS structure could interfere with metabolic co-ordination between the C3 and C4 cycles, decrease metabolite flow rate between the tissues, increase CO2 leakage from the bundle sheath, and slow enzyme activity. C4 plants have substantial acclimation potential, but in most cases lag behind the acclimation responses in C3 plants. For example, some C4 species are unable to maintain high quantum yields when grown in low-light conditions. Others fail to reduce carboxylase content in shade, leaving substantial over-capacity of Rubisco and PEP carboxylase in place. Shade-tolerant C4 grasses lack the capacity for maintaining a high state of photosynthetic induction following sunflecks, and thus may be poorly suited to exploit subsequent sunflecks compared with C3 species. In total, the evidence indicates that C4 photosynthesis is less phenotypically plastic than C3 photosynthesis, and this may contribute to the more restricted ecological and geographical distribution of C4 plants across the Earth.

The temperature response of net CO(2) assimilation rate (A), the rate of whole-chain electron transport, the activity and activation state of Rubisco, and the pool sizes of ribulose-1,5-bisphosphate (RuBP) and 3-phosphoglyceric acid (PGA) were assessed in sweet potato (Ipomoea batatas) grown under greenhouse conditions. Above the thermal optimum of photosynthesis, the activation state of Rubisco declined with increasing temperature. Doubling CO(2) above 370 mubar further reduced the activation state, while reducing CO(2) by one-half increased it. At cool temperature (<16 degrees C), the activation state of Rubisco declined at CO(2) levels where photosynthesis was unaffected by a 90% reduction in O(2) content. Reduction of the partial pressure of CO(2) at cool temperature also enhanced the activation state of Rubisco. The rate of electron transport showed a pronounced temperature response with the same temperature optimum as A at elevated CO(2). RuBP pool size and the RuBP-to-PGA ratio declined with increasing temperature. Increasing CO(2) also reduced the RuBP pool size. These results are consistent with the hypothesis that the reduction in the activation state of Rubisco at high and low temperature is a regulated response to a limitation in one of the processes contributing to the rate of RuBP regeneration. To further evaluate this possibility, we used measured estimates of Rubisco capacity, electron transport capacity, and the inorganic phosphate regeneration capacity to model the response of A to temperature. At elevated CO(2), the activation state of Rubisco declined at high temperatures where electron transport capacity was predicted to be limiting, and at cooler temperatures where the inorganic phosphate regeneration capacity was limiting. At low CO(2), where Rubisco capacity was predicted to limit photosynthesis, full activation of Rubisco was observed at all measurement temperatures.

Crassulacean acid metabolism (CAM) has evolved independently on dozens of occasions and is now found in over 7% of plant species. In this study, the leaf structure of a phylogenetically diverse assemblage of 18 CAM plants was compared with six C 3 plants and four C 4 plants to assess whether consistent anatomical patterns that may reflect functional constraints are present. CAM plants exhibited increased cell size and increased leaf and mesophyll thickness relative to C 3 and C 4 species. CAM species also exhibited reduced intercellular air space (IAS) and reduced length of mesophyll surface exposed to IAS per unit area (L mes  / area). The low volume of IAS and low exposure of mesophyll surface to IAS likely increases internal resistance to CO 2 in CAM tissues. While this diffusional barrier may limit uptake of CO 2 during Phases II and IV, carbon economy could be enhanced through the reduced loss of internal CO 2 during all four phases of CAM.

Xylem structure and function is proposed to reflect an evolutionary balance between demands for efficient movement of water to the leaf canopy and resistance to cavitation during high xylem tension. Water use efficiency (WUE) affects this balance by altering the water cost of photosynthesis. Therefore species of greater WUE, such as C(4) plants, should have altered xylem properties. To evaluate this hypothesis, we assessed the hydraulic and anatomical properties of 19 C(3) and C(4) woody species from arid regions of the American west and central Asia. Specific conductivity of stem xylem ( K(s) ) was 16%-98% lower in the C(4) than C(3) shrubs from the American west. In the Asian species, the C(3) Nitraria schoberi had similar and Halimodendron halodendron higher K(s) values compared with three C(4) species. Leaf specific conductivity ( K(L); hydraulic conductivity per leaf area) was 60%-98% lower in the C(4) than C(3) species, demonstrating that the presence of the C(4) pathway alters the relationship between leaf area and the ability of the xylem to transport water. C(4) species produced similar or smaller vessels than the C(3) shrubs except in Calligonum, and most C(4) shrubs exhibited higher wood densities than the C(3) species. Together, smaller conduit size and higher wood density indicate that in most cases, the C(4) shrubs exploited higher WUE by altering xylem structure to enhance safety from cavitation. In a minority of cases, the C(4) shrubs maintained similar xylem properties but enhanced the canopy area per branch. By establishing a link between C(4) photosynthesis and xylem structure, this study indicates that other phenomena that affect WUE, such as atmospheric CO(2) variation, may also affect the evolution of wood structure and function.

C 4 photosynthesis is a series of anatomical and biochemical modifications that concentrate CO 2 around the carboxylating enzyme Rubisco, thereby increasing photosynthetic efficiency in conditions promoting high rates of photorespiration. The C 4 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 C 4 photosynthesis occurred in the dicots, with at least 30 lineages. C 4 photosynthesis first arose in grasses, probably during the Oligocene epoch (24-35 million yr ago). The earliest C 4 dicots are likely members of the Chenopodiaceae dating back 15-21 million yr; however, most C 4 dicot lineages are estimated to have appeared relatively recently, perhaps less than 5 million yr ago. C 4 photosynthesis in the dicots originated in arid regions of low latitude, implicating combined effects of heat, drought and/or salinity as important conditions promoting C 4 evolution. Low atmospheric CO 2 is a significant contributing factor, because it is required for high rates of photorespiration. Consistently, the appearance of C 4 plants in the evolutionary record coincides with periods of increasing global aridification and declining atmospheric CO 2 . Gene duplication followed by neo- and nonfunctionalization are the leading mechanisms for creating C 4 genomes, with selection for carbon conservation traits under conditions promoting high photorespiration being the ultimate factor behind the origin of C 4 photosynthesis. Contents Summary 341 I. Introduction 342 II. What is C 4 photosynthesis? 343 III. Why did C 4 photosynthesis evolve? 347 IV. Evolutionary lineages of C 4 photosynthesis 348 V. Where did C 4 photosynthesis evolve? 350 VI. How did C 4 photosynthesis evolve? 352 VII. Molecular evolution of C 4 photosynthesis 361 VIII. When did C 4 photosynthesis evolve 362 IX. The rise of C 4 photosynthesis in relation to climate and CO 2 363 X. Final thoughts: the future evolution of C 4 photosynthesis 365 Acknowledgements 365 References 365.

C(4) plants are rare in cool climates, an ecological pattern attributable to their poor photosynthetic performance at low temperatures relative to C(3) species. However, some C(4) species are able to persist at high latitudes and high elevations, possibly due to the characteristics of the particular microsites they inhabit in these otherwise unfavourable environments. One such species is Muhlenbergia glomerata, which occurs above 60 degrees N in Canada and is found in the atypical C(4) habitat of boreal fens. In this study, we evaluate how microsite features affect the success of M. glomerata in boreal fens. We surveyed 19 populations across northern Ontario during the summers of 1999 and 2000. The ground coverage by woody vegetation was the most important parameter affecting the presence or absence of M. glomerata. Woody plants covered over 50% of the ground area in plots where M. glomerata is absent, but less than 20% where it is present. The minimum light intensity threshold for the presence of the C(4) species was about 32% of full-sunlight at plant height. Surprisingly, in boreal fens M. glomerata was largely restricted to the wetter moss hollows, rather than occurring on the dry hummocks where its greater water use efficiency might have been advantageous. Woody species dominated the hummocks, but were uncommon in the hollows. In these cool northern climates M. glomerata apparently persists because sufficient periods of temperatures favourable to C(4) photosynthesis occur, but this persistence likely requires some factor that suppresses the woody vegetation.

C(4) plants are rare in the cool climates characteristic of high latitudes and elevations, but the reasons for this are unclear. We tested the hypothesis that CO(2) fixation by Rubisco is the rate-limiting step during C(4) photosynthesis at cool temperatures. We measured photosynthesis and chlorophyll fluorescence from 6 degrees C to 40 degrees C, and in vitro Rubisco and phosphoenolpyruvate carboxylase activity from 0 degrees C to 42 degrees C, in Flaveria bidentis modified by an antisense construct (targeted to the nuclear-encoded small subunit of Rubisco, anti-RbcS) to have 49% and 32% of the wild-type Rubisco content. Photosynthesis was reduced at all temperatures in the anti-Rbcs plants, but the thermal optimum for photosynthesis (35 degrees C) did not differ. The in vitro turnover rate (kcat) of fully carbamylated Rubisco was 3.8 mol mol(-)(1) s(-)(1) at 24 degrees C, regardless of genotype. The in vitro kcat (Rubisco Vcmax per catalytic site) and in vivo kcat (gross photosynthesis per Rubisco catalytic site) were the same below 20 degrees C, but at warmer temperatures, the in vitro capacity of the enzyme exceeded the realized rate of photosynthesis. The quantum requirement of CO(2) assimilation increased below 25 degrees C in all genotypes, suggesting greater leakage of CO(2) from the bundle sheath. The Rubisco flux control coefficient was 0.68 at the thermal optimum and increased to 0.99 at 6 degrees C. Our results thus demonstrate that Rubisco capacity is a principle control over the rate of C(4) photosynthesis at low temperatures. On the basis of these results, we propose that the lack of C(4) success in cool climates reflects a constraint imposed by having less Rubisco than their C(3) competitors.

C(4) plants are directly affected by all major global change parameters, often in a manner that is distinct from that of C(3) plants. Rising CO(2) generally stimulates C(3) photosynthesis more than C(4), but C(4) species still exhibit positive responses, particularly at elevated temperature and arid conditions where they are currently common. Acclimation of photosynthesis to high CO(2) occurs in both C(3) and C(4) plants, most notably in nutrient-limited situations. High CO(2) aggravates nitrogen limitations and in doing so may favor C(4) species, which have greater photosynthetic nitrogen use efficiency. C(4) photosynthesis is favored by high temperature, but global warming will not necessarily favor C(4) over C(3) plants because the timing of warming could be more critical than the warming itself. C(3) species will likely be favored where harsh winter climates are moderated, particularly where hot summers also become drier and less favorable to C(4) plant growth. Eutrophication of soils by nitrogen deposition generally favors C(3) species by offsetting the superior nitrogen use efficiency of C(4) species; this should allow C(3) species to expand at the expense of C(4) plants. Land-use change and biotic invasions are also important global change factors that affect the future of C(4) plants. Human exploitation of forested landscapes favors C(4) species at low latitude by removing woody competitors and opening gaps in which C(4) grasses can establish. Invasive C(4) grasses are causing widespread forest loss in Asia, the Americas and Oceania by accelerating fire cycles and reducing soil nutrient status. Once established, weedy C(4) grasses can prevent woodland establishment, and thus arrest ecological succession. In sum, in the future, certain C(4) plants will prosper at the expense of C(3) species, and should be able to adjust to the changes the future brings.

C 4 plants are uncommon in cold environments and do not generally occur in the alpine tundra. In the White Mountains of California, however, the C 4 grass Muhlenbergia richardsonis is common in the alpine zone at 3,300-3,800 m, with the highest population observed at 3,960 m (13,000 feet) above sea level. This is the highest reported C 4 species in North America and is near the world altitude limit for C 4 plants (4,000-4,500 m). Above 3,800 m, M. richardsonis is largely restricted to southern slope aspects, with greatest frequency on southeast-facing slopes. In open tundra, M. richardsonis formed prostrate mats with a mean height of 2.5 cm. Neighboring C 3 grasses were two to three times taller. Because of its short stature, leaf temperature of M. richardsonis was greatly influenced by the boundary layer of the ground, rising over 20°C above air temperature in full sun and still air and over 10°C above air temperature in full sun and wind velocity of 1-4 m s -1 . Thus, although air temperatures did not exceed 15°C, midday leaf temperatures of M. richardsonis were routinely between 25°C and 35°C, a range favorable to C 4 photosynthesis. At night, leaf temperature of M. richardsonis was often 5-12°C below air temperature, resulting in regular exposure to subzero temperatures and frosting of the leaves. No visible injury was associated with exposure to freezing night temperatures. The presence of M. richardsonis in the alpine zone demonstrates that C 4 plants can tolerate extreme cold during the growing season. The localization to microsites where leaf temperatures can exceed 25°C during the day, however, indicates that even when cold tolerant, C 4 plants still require periods of high leaf temperature to remain competitive with C 3 species. In this regard, the prostrate growth form of M. richardsonis compensates for the alpine climate by allowing sufficient heating of the leaf canopy during the day.

C(4) photosynthesis in terrestrial plants was thought to require Kranz anatomy because the cell wall between mesophyll and bundle sheath cells restricts leakage of CO(2). Recent work with the central Asian chenopods Borszczowia aralocaspica and Bienertia cycloptera show that C(4) photosynthesis functions efficiently in individual cells containing both the C(4) and C(3) cycles. These discoveries provide new inspiration for efforts to convert C(3) crops into C(4) plants because the anatomical changes required for C(4) photosynthesis might be less stringent than previously thought.

Because of anthropogenic increases in atmospheric CO(2) content, there is a need to understand how organisms sense and respond to CO(2) variation. An important distinction is whether CO(2) responses result from direct effects of CO(2) on signal-transduction pathways, enzyme catalysis, or regulatory processes, as opposed to indirect, secondary responses that are a consequence of the direct effects. In plants, direct effects occur because rising CO(2) A) increases the activity of Ribulose-1,5-bisphopshate carboxylase/oxygenase (Rubisco) via its role as a substrate for RuBP carboxylation and its inhibition of RuBP oxygenation; B) reduces stomatal aperture; C) alters mitochondrial respiration; and D) possibly reduces transcription of genes for Rubisco activase and carbonic anhydrase. Because of these direct effects, the carbon and water balance of plants is altered leading to secondary effects on growth, resource partitioning and defense compound synthesis. Reduced investment in photosynthetic protein is one of the characteristic acclimation responses of plants to high CO(2). This is modulated by increased carbohydrate levels, probably in concert with hormone signals from the roots. Roots are hypothesized to be the main control points for CO(2) acclimation because they are well situated to integrate the carbohydrate status of the plant. In higher fungi, development of the mushroom fruiting body is inhibited at high CO(2), but the mechanism is poorly known. Fungal CO(2) sensing may serve to position the spore-bearing tissue above the soil boundary layer to ensure effective spore dispersal. The animals that are most sensitive to anthropogenic CO(2) enrichment are insects. Many insects have a well-developed ability to sense CO(2) variation as a means of locating food. Unlike plants, insects have CO(2) receptors that can detect variation in CO(2) as low as 0.5 ppm. However, the sensitivity of these receptors is reduced in atmospheres with double or triple current levels of CO(2), indicating some insect species may be threatened by rising atmospheric CO(2).

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. Despite sharing a similar metabolism, crassulacean acid metabolism (CAM) and C4 photosynthesis are not known to occur in identical species, with the exception of Portulaca spp. In Portulaca, C4 and weak CAM photosynthesis occur in distinct regions of the leaf, rather than in the same cells. This is in marked contrast to the situation in most CAM species where C3 and CAM photosynthesis are active in the same cell over the course of a day and growing season. The lack of CAM and C4 photosynthesis in identical cells of a plant indicates these photosynthetic pathways are incompatible. Incompatibilities between CAM and C4 photosynthesis could have a number of biochemical, anatomical and evolutionary explanations. Biochemical incompatibilities could result from the requirement for spatial separation of C3 and C4 phases in C4 plants versus temporal separation in CAM plants. In C4 plants, regulatory systems coordinate mesophyll and bundle sheath metabolism, with light intensity being the major environmental signal. In CAM plants, a circadian oscillator coordinates day and night phases of CAM. The requirement for rapid intercellular transport in C4 plants may be incompatible with the intracellular transport and storage needs of CAM. For example, the large vacuole required for malate storage in CAM could impede metabolite diffusion between mesophyll and bundle sheath cells in C4 plants. Anatomical barriers could also exist because both CAM and the C4 pathway require distinct leaf anatomies for efficient function. Efficient function of the C4 pathway generally requires an outer layer of cells specialized for phosphoenolpyruvate (PEP) carboxylation and regeneration and an inner layer for CO2 accumulation and refixation, while CAM species require enlarged vacuoles and tight cell packing. In evolutionary terms, barriers preventing CAM and C4 photosynthesis in the same species may be the initial steps in the respective evolutionary pathways from C3 ancestors. The first steps in C4 photosynthesis are related to scavenging photorespiratory CO2 via localization of glycine decarboxylase in the bundle sheath cells. The initial steps in CAM evolution are associated with the scavenging of respiratory CO2 at night by PEP carboxylation. In each, simplified versions of the specialized anatomy may need to be present for the evolutionary sequence to begin. For C4 evolution, enhanced bundle sheath size may be required in C3 ancestors; for CAM evolution, succulence may be required. Thus, before CAM or C4 photosynthesis began to evolve, the outcome of the evolutionary experiment may have been predetermined.

The capacity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to consume RuBP is a major limitation on the rate of net CO(2) assimilation (A) in C(3) and C(4) plants. The pattern of Rubisco limitation differs between the two photosynthetic types, as shown by comparisons of temperature and CO(2) responses of A and Rubisco activity from C(3) and C(4) species. In C(3) species, Rubisco capacity is the primary limitation on A at light saturation and CO(2) concentrations below the current atmospheric value of 37 Pa, particularly near the temperature optimum. Below 20 degrees C, C(3) photosynthesis at 37 and 68 Pa is often limited by the capacity to regenerate phosphate for photophosphorylation. In C(4) plants, the Rubisco capacity is equivalent to A below 18 degrees C, but exceeds the photosynthetic capacity above 25 degrees C, indicating that Rubisco is an important limitation at cool but not warm temperatures. A comparison of the catalytic efficiency of Rubisco (k(cat) in mol CO(2) mol(-1) Rubisco active sites s(-1)) from 17 C(3) and C(4) plants showed that Rubisco from C(4) species, and C(3) species originating in cool environments, had higher k(cat) than Rubisco from C(3) species originating in warm environments. This indicates that Rubisco evolved to improve performance in the environment that plants normally experience. In C(4) plants, and C(3) species from cool environments, Rubisco often operates near CO(2) saturation, so that increases in k(cat) would enhance A. In warm-habitat C(4) species, Rubisco often operates at CO(2) concentrations below the K(m) for CO(2). Because k(cat) and K(m) vary proportionally, the low k(cat) indicates that Rubisco has been modified in a manner that reduces K(m) and thus increases the affinity for CO(2) in C(3) species from warm climates.

Using gas exchange, enzyme assays, and theoretical modeling of photosynthetic responses to light and CO(2), we investigated whether decarbamylation of the active site of Rubisco at low CO(2) and low light leads to a condition where the activation state of Rubisco directly limits the rate of net CO(2) assimilation. Photosynthetic limitation by a reduction in the activation state of Rubisco would be indicated as a decline in the initial slope of the photosynthetic CO(2) response relative to what is predicted using theoretical models. In bean (Phaseolus vulgaris) and oat (Avena sativa), we saw no discrepancy between predicted and observed initial slope values at 200 and 400 mbar O(2), indicating no limitation by the carbamylation state of Rubisco. At 30 mbar O(2) and light saturation, we also saw no discrepancy between predicted and observed initial slope values; however, at subsaturating light intensity, our observed initial slope values were less than the modeled initial slope values that corresponded to an RuBP regeneration limitation. Moreover, significant reduction of the Rubisco activation state occurred in both species at 30 mbar O(2) and 30 mubar CO(2). When the model was reprogrammed to account for observed levels of Rubisco deactivation, the predicted and measured initial slope values at low O(2) and low PPFD were similar, indicating the reduction in carbamylation state accounted for the discrepancy. We interpret this as evidence for a direct limitation of the carbamylation state of Rubisco, probably because of a CO(2) limitation for carbamate formation. This limitation was only observed at intercellular CO(2) levels below what is encountered in vivo. At physiologically relevant CO(2) levels in situ, the leaves maintained sufficient Rubisco activity to avoid cabamylation state limitations in the steady state.