Stomatal response to VPD in C4 plants with different biochemical sub-pathways.

C4 NAD-malic enzyme (NAD-ME) species occurs in drier regions and exhibit different drought responses compared to C4 NADP-malic enzyme (NADP-ME) species. However, a physiological mechanism explaining the geographical discrepancies remains uncertain. This study examined gas exchange patterns that might explain different distributions observed between two subtypes of C4 photosynthesis. We measured the response of leaf gas exchange to vapour pressure deficit (VPD) and CO2 in plants from six distinct C4 clades having closely related NAD-ME and NADP-ME species using a Li-Cor 6400 gas exchange system. We found that NAD-ME species exhibited greater relative reductions in stomatal conductance with increases in VPD than NADP-ME species but observed no consistent subtype differences in C4 cycle activity as indicated by the initial slope of the A response to intercellular CO2 concentration. Based on these results, we hypothesise the greater response of gs to increasing VPD may enable NAD-ME plants to outperform NADP-ME plants in hot, dry environments where VPD is normally high.

Increased α-ketoglutarate links the C3-C4 intermediate state to C4 photosynthesis in the genus Flaveria.

As a complex trait, C4 photosynthesis has multiple independent origins in evolution. Phylogenetic evidence and theoretical analysis suggest that C2 photosynthesis, which is driven by glycine decarboxylation in the bundle sheath cell, may function as a bridge from C3 to C4 photosynthesis. However, the exact molecular mechanism underlying the transition between C2 photosynthesis to C4 photosynthesis remains elusive. Here, we provide evidence suggesting a role of higher α-ketoglutarate (AKG) concentration during this transition. Metabolomic data of 12 Flaveria species, including multiple photosynthetic types, show that AKG concentration initially increased in the C3-C4 intermediate with a further increase in C4 species. Petiole feeding of AKG increases the concentrations of C4-related metabolites in C3-C4 and C4 species but not the activity of C4-related enzymes. Sequence analysis shows that glutamate synthase (Fd-GOGAT), which catalyzes the generation of glutamate using AKG, was under strong positive selection during the evolution of C4 photosynthesis. Simulations with a constraint-based model for C3-C4 intermediate further show that decreasing the activity of Fd-GOGAT facilitated the transition from a C2-dominant to a C4-dominant CO2 concentrating mechanism. All these results provide insight into the mechanistic switch from C3-C4 intermediate to C4 photosynthesis.

The CAM lineages of planet Earth.

BACKGROUND AND SCOPE: The growth of experimental studies of crassulacean acid metabolism (CAM) in diverse plant clades, coupled with recent advances in molecular systematics, presents an opportunity to re-assess the phylogenetic distribution and diversity of species capable of CAM. It has been more than two decades since the last comprehensive lists of CAM taxa were published, and an updated survey of the occurrence and distribution of CAM taxa is needed to facilitate and guide future CAM research. We aimed to survey the phylogenetic distribution of these taxa, their diverse morphology, physiology and ecology, and the likely number of evolutionary origins of CAM based on currently known lineages. RESULTS AND CONCLUSIONS: We found direct evidence (in the form of experimental or field observations of gas exchange, day-night fluctuations in organic acids, carbon isotope ratios and enzymatic activity) for CAM in 370 genera of vascular plants, representing 38 families. Further assumptions about the frequency of CAM species in CAM clades and the distribution of CAM in the Cactaceae and Crassulaceae bring the currently estimated number of CAM-capable species to nearly 7 % of all vascular plants. The phylogenetic distribution of these taxa suggests a minimum of 66 independent origins of CAM in vascular plants, possibly with dozens more. To achieve further insight into CAM origins, there is a need for more extensive and systematic surveys of previously unstudied lineages, particularly in living material to identify low-level CAM activity, and for denser sampling to increase phylogenetic resolution in CAM-evolving clades. This should allow further progress in understanding the functional significance of this pathway by integration with studies on the evolution and genomics of CAM in its many forms.

CAM photosynthesis in Bulnesia retama (Zygophyllaceae), a non-succulent desert shrub from South America.

BACKGROUND AND AIMS: Bulnesia retama is a drought-deciduous, xerophytic shrub from arid landscapes of South America. In a survey of carbon isotope ratios (δ13C) in specimens from the field, B. retama exhibited less negative values, indicative of CAM or C4 photosynthesis. Here, we investigate whether B. retama is a C4 or CAM plant. METHODS: Gas-exchange responses to intercellular CO2, diurnal gas-exchange profiles, δ13C and dawn vs. afternoon titratable acidity were measured on leaves and stems of watered and droughted B. retama plants. Leaf and stem cross-sections were imaged to determine whether the tissues exhibited succulent CAM or C4 Kranz anatomy. KEY RESULTS: Field-collected stems and fruits of B. retama exhibited δ13C between -16 and -19 ‰. Plants grown in a glasshouse from field-collected seeds had leaf δ13C values near -31 ‰ and stem δ13C values near -28 ‰. The CO2 response of photosynthesis showed that leaves and stems used C3 photosynthesis during the day, while curvature in the nocturnal response of net CO2 assimilation rate (A) in all stems, coupled with slightly positive rates of A at night, indicated modest CAM function. C4 photosynthesis was absent. Succulence was absent in all tissues, although stems exhibited tight packing of the cortical chlorenchyma in a CAM-like manner. Tissue titratable acidity increased at night in droughted stems. CONCLUSIONS: Bulnesia retama is a weak to modest C3 + CAM plant. This is the first report of CAM in the Zygophyllaceae and the first showing that non-succulent, xerophytic shrubs use CAM. CAM alone in B. retama was too limited to explain less negative δ13C in field-collected plants, but combined with effects of low stomatal and mesophyll conductance it could raise δ13C to observed values between -16 and -19 ‰. Modest CAM activity, particularly during severe drought, could enable B. retama to persist in arid habitats of South America.

Atmospheric CO2 decline and the timing of CAM plant evolution.

BACKGROUND AND AIMS: CAM photosynthesis is hypothesized to have evolved in atmospheres of low CO2 concentration in recent geological time because of its ability to concentrate CO2 around Rubisco and boost water use efficiency relative to C3 photosynthesis. We assess this hypothesis by compiling estimates of when CAM clades arose using phylogenetic chronograms for 73 CAM clades. We further consider evidence of how atmospheric CO2 affects CAM relative to C3 photosynthesis. RESULTS: Where CAM origins can be inferred, strong CAM is estimated to have appeared in the past 30 million years in 46 of 48 examined clades, after atmospheric CO2 had declined from high (near 800 ppm) to lower (<450 ppm) values. In turn, 21 of 25 clades containing CAM species (but where CAM origins are less certain) also arose in the past 30 million years. In these clades, CAM is probably younger than the clade origin. We found evidence for repeated weak CAM evolution during the higher CO2 conditions before 30 million years ago, and possible strong CAM origins in the Crassulaceae during the Cretaceous period prior to atmospheric CO2 decline. Most CAM-specific clades arose in the past 15 million years, in a similar pattern observed for origins of C4 clades. CONCLUSIONS: The evidence indicates strong CAM repeatedly evolved in reduced CO2 conditions of the past 30 million years. Weaker CAM can pre-date low CO2 and, in the Crassulaceae, strong CAM may also have arisen in water-limited microsites under relatively high CO2. Experimental evidence from extant CAM species demonstrates that elevated CO2 reduces the importance of nocturnal CO2 fixation by increasing the contribution of C3 photosynthesis to daily carbon gain. Thus, the advantage of strong CAM would be reduced in high CO2, such that its evolution appears less likely and restricted to more extreme environments than possible in low CO2.

The Evolution of C4 Photosynthesis in Flaveria (Asteraceae): Insights from the Flaveria linearis Complex.

Flaveria is a leading model for C4 plant evolution due to the presence of a dozen C3-C4 intermediate species, many of which are associated with a phylogenetic complex centered around Flaveria linearis. To investigate C4 evolution in Flaveria, we updated the Flaveria phylogeny and evaluated gas exchange, starch δ13C, and activity of C4 cycle enzymes in 19 Flaveria species and 28 populations within the F. linearis complex. A principal component analysis identified six functional clusters: (1) C3, (2) sub-C2, (3) full C2, (4) enriched C2, (5) sub-C4, and (6) fully C4 species. The sub-C2 species lacked a functional C4 cycle, while a gradient was present in the C2 clusters from little to modest C4 cycle activity as indicated by δ13C and enzyme activities. Three Yucatan populations of F. linearis had photosynthetic CO2 compensation points equivalent to C4 plants but showed little evidence for an enhanced C4 cycle, indicating they have an optimized C2 pathway that recaptures all photorespired CO2 in the bundle sheath (BS) tissue. All C2 species had enhanced aspartate aminotransferase activity relative to C3 species and most had enhanced alanine aminotransferase activity. These aminotransferases form aspartate and alanine from glutamate and in doing so could help return photorespiratory nitrogen (N) from BS to mesophyll cells, preventing glutamate feedback onto photorespiratory N assimilation. Their use requires upregulation of parts of the C4 metabolic cycle to generate carbon skeletons to sustain N return to the mesophyll, and thus could facilitate the evolution of the full C4 photosynthetic pathway.

Seed size effects on plant establishment under low atmospheric CO2, with implications for seed size evolution.

BACKGROUND AND AIMS: Low atmospheric CO2 concentration depresses photosynthesis and resource use efficiency, and therefore can inhibit phases of the life cycle such as seedling establishment. Seed reserves can compensate for photosynthetic inhibition by accelerating seedling growth. We therefore hypothesize that seedlings arising from large seeds show less inhibition from low atmospheric CO2 than young plants from small seeds. Seed size effects on seedling responses to low CO2 may also be enhanced in warm environments, due to greater photorespiration at high temperature. METHODS: Phaseolus and Vigna seeds differing in mass by over two orders of magnitude were planted and grown for 14 d in growth chambers with CO2 concentrations of 370, 180 or 100 ppm, in thermal regimes of 25 °C/19 °C, 30 °C/24 °C or 35 °C/29 °C (day/night). We measured leaf area expansion, shoot growth and mortality of the seedlings arising from the variously sized seeds at 14 days after planting (14 DAP). KEY RESULTS: Relative to small-seeded plants, large-seeded genotypes produced greater leaf area and shoot mass at 14 DAP across the range of CO2 treatments in the 25 °C/19 °C and 30 °C/24 °C regimes, and at 100 ppm in the 35 °C/29 °C treatment. The proportional decline in leaf area and seed mass with CO2 reduction was generally greater for seedlings arising from small than from large seeds. Reductions in leaf area due to CO2 reduction increased in the warmer temperature treatments. In the 35 °C/19 °C treatment at 100 ppm CO2, seedling mortality was greater in small- than in large-seeded genotypes, and the small-seeded genotypes were unable to exit the seedling stage by the end of the experiment. CONCLUSIONS: The results support a hypothesis that seedlings from large seeds grow and establish better than seedlings from small seeds in warm, low CO2 environments. During low CO2 episodes in Earth's history, such as the past 30 million years, large seeds may have been favoured by natural selection in warm environments. With the recent rise in atmospheric CO2 due to human activities, trade-offs between seed size and number may already be affected, such that seed size today may be non-optimal in their natural habitats.

Plants and water: the search for a comprehensive understanding.

We learn early in life sciences classes that water is the solution of life, working in tandem with carbon to make life as we know it possible. Globally, the abundance of water can be misleading, as most of this water is unavailable, being overly salinized in the oceans or locked in deep underground reserves. On land, the critical supply is of freshwater, which is unevenly distributed in space and time. Even the wettest environments can experience episodic water deficit, and flash flooding periodically occurs in arid landscapes. While humanity can capture, store and transport freshwater over large distances to ensure sustained supply, such options are not apparent for plants except in an immediate local context. Plants must make do with the water in their immediate surroundings, whether it be abundant or scarce. How they do this has led to a myriad of adaptive solutions, involving capturing, storing and transporting water. The traits that enable them to optimize water use in a range of hydraulic environments, subject to multivariate selective constraints, are the essence of the discipline of plant-water relations.

The crucial roles of mitochondria in supporting C4 photosynthesis.

C4 photosynthesis involves a series of biochemical and anatomical traits that significantly improve plant productivity under conditions that reduce the efficiency of C3 photosynthesis. We explore how evolution of the three classical biochemical types of C4 photosynthesis (NADP-ME, NAD-ME and PCK types) has affected the functions and properties of mitochondria. Mitochondria in C4 NAD-ME and PCK types play a direct role in decarboxylation of metabolites for C4 photosynthesis. Mitochondria in C4 PCK type also provide ATP for C4 metabolism, although this role for ATP provision is not seen in NAD-ME type. Such involvement has increased mitochondrial abundance/size and associated enzymatic capacity, led to changes in mitochondrial location and ultrastructure, and altered the role of mitochondria in cellular carbon metabolism in the NAD-ME and PCK types. By contrast, these changes in mitochondrial properties are absent in the C4 NADP-ME type and C3 leaves, where mitochondria play no direct role in photosynthesis. From an eco-physiological perspective, rates of leaf respiration in darkness vary considerably among C4 species but does not differ systematically among the three C4 types. This review outlines further mitochondrial research in key areas central to the engineering of the C4 pathway into C3 plants and to the understanding of variation in rates of C4 dark respiration.

Digest: Between invasive species and a hot place: Plant evolution under climate change.

Will climate change lead to invasive species evolving faster than native or naturalized species? Gianoli and Molina-Montenegro showed that, under warming and drought, the evolution of photosynthetic capacity does not always favor invasive species. These data raise interesting questions for the study of evolution of invasive species under climate change.

The coordination of major events in C4 photosynthesis evolution in the genus Flaveria.

C4 photosynthesis is a remarkable complex trait, elucidations of the evolutionary trajectory of C4 photosynthesis from its ancestral C3 pathway can help us better understand the generic principles of the evolution of complex traits and guide the engineering of C3 crops for higher yields. Here, we used the genus Flaveria that contains C3, C3-C4, C4-like and C4 species as a system to study the evolution of C4 photosynthesis. We first mapped transcript abundance, protein sequence and morphological features onto the phylogenetic tree of the genus Flaveria, and calculated the evolutionary correlation of different features; we then predicted the relative changes of ancestral nodes of those features to illustrate the major events during the evolution of C4 photosynthesis. We found that gene expression and protein sequence showed consistent modification patterns in the phylogenetic tree. High correlation coefficients ranging from 0.46 to 0.9 among gene expression, protein sequence and morphology were observed. The greatest modification of those different features consistently occurred at the transition between C3-C4 species and C4-like species. Our results show highly coordinated changes in gene expression, protein sequence and morphological features, which support evolutionary major events during the evolution of C4 metabolism.

Russ Monson and the evolution of C4 photosynthesis.

Early in his career, Russ Monson produced a series of influential eco-physiological papers that helped lay the foundation for the study of C4 plant evolution. Among the most important was a 1984 paper with Maurice Ku and Gerry Edwards that outlined the pathway for the evolutionary bridge from C3 to C4 photosynthesis. This model proposed C4 photosynthesis arose out of a shuttle that imported photorespiratory metabolites into bundle sheath (BS) cells, where glycine decarboxylase cleaved off CO2, allowing it to accumulate and be efficiently refixed by BS Rubisco. By the mid-1990's, Monson's research focus had shifted away from C4 plants, save for one 2003 paper on C3 versus C4 stomatal control with Travis Huxman, and a series of critical reviews on C4 evolution. These reviews heavily influenced the modern synthesis of C4 evolutionary studies, which incorporates phylogenomic understanding with physiological, molecular, and structural characterizations of trait shifts in multiple evolutionary lineages. Subsequent research supported the Monson et al. model from 1984, by showing a glycine shuttle occurs in nearly all C3-C4 intermediate species identified. Monson also examined the physiological controls over the ecological distribution of C3, C3-C4 intermediate, and C4 photosynthesis, building our understanding of the fitness value of the intermediate and C4 pathway in relevant microenvironments. By establishing the foundation for discoveries that followed, Russ Monson can rightly be considered a leading pioneer contributing to the evolutionary biology of C4 photosynthesis.

Evolutionary Convergence of C4 Photosynthesis: A Case Study in the Nyctaginaceae.

C4 photosynthesis evolved over 65 times, with around 24 origins in the eudicot order Caryophyllales. In the Caryophyllales family Nyctaginaceae, the C4 pathway is known in three genera of the tribe Nyctagineae: Allionia, Okenia and Boerhavia. Phylogenetically, Allionia and Boerhavia/Okenia are separated by three genera whose photosynthetic pathway is uncertain. To clarify the distribution of photosynthetic pathways in the Nyctaginaceae, we surveyed carbon isotope ratios of 159 species of the Nyctaginaceae, along with bundle sheath (BS) cell ultrastructure, leaf gas exchange, and C4 pathway biochemistry in five species from the two C4 clades and closely related C3 genera. All species in Allionia, Okenia and Boerhavia are C4, while no C4 species occur in any other genera of the family, including three that branch between Allionia and Boerhavia. This demonstrates that C4 photosynthesis evolved twice in Nyctaginaceae. Boerhavia species use the NADP-malic enzyme (NADP-ME) subtype of C4 photosynthesis, while Allionia species use the NAD-malic enzyme (NAD-ME) subtype. The BS cells of Allionia have many more mitochondria than the BS of Boerhavia. Bundle sheath mitochondria are closely associated with chloroplasts in Allionia which facilitates CO2 refixation following decarboxylation by mitochondrial NAD-ME. The close relationship between Allionia and Boerhavia could provide insights into why NADP-ME versus NAD-ME subtypes evolve, particularly when coupled to analysis of their respective genomes. As such, the group is an excellent system to dissect the organizational hierarchy of convergent versus divergent traits produced by C4 evolution, enabling us to understand when convergence is favored versus when divergent modifications can result in a common phenotype.

Global change biology: A primer.

Because of human action, the Earth has entered an era where profound changes in the global environment are creating novel conditions that will be discernable far into the future. One consequence may be a large reduction of the Earth's biodiversity, potentially representing a sixth mass extinction. With effective stewardship, the global change drivers that threaten the Earth's biota could be alleviated, but this requires clear understanding of the drivers, their interactions, and how they impact ecological communities. This review identifies 10 anthropogenic global change drivers and discusses how six of the drivers (atmospheric CO2 enrichment, climate change, land transformation, species exploitation, exotic species invasions, eutrophication) impact Earth's biodiversity. Driver impacts on a particular species could be positive or negative. In either case, they initiate secondary responses that cascade along ecological lines of connection and in doing so magnify the initial impact. The unique nature of the threat to the Earth's biodiversity is not simply due to the magnitude of each driver, but due to the speed of change, the novelty of the drivers, and their interactions. Emphasizing one driver, notably climate change, is problematic because the other global change drivers also degrade biodiversity and together threaten the stability of the biosphere. As the main academic journal addressing global change effects on living systems, GCB is well positioned to provide leadership in solving the global change challenge. If humanity cannot meet the challenge, then GCB is positioned to serve as a leading chronicle of the sixth mass extinction to occur on planet Earth.

The Evolutionary Origin of C4 Photosynthesis in the Grass Subtribe Neurachninae.

The Australian grass subtribe Neurachninae contains closely related species that use C3, C4, and C2 photosynthesis. To gain insight into the evolution of C4 photosynthesis in grasses, we examined leaf gas exchange, anatomy and ultrastructure, and tissue localization of Gly decarboxylase subunit P (GLDP) in nine Neurachninae species. We identified previously unrecognized variation in leaf structure and physiology within Neurachne that represents varying degrees of C3-C4 intermediacy in the Neurachninae. These include inverse correlations between the apparent photosynthetic carbon dioxide (CO2) compensation point in the absence of day respiration (C * ) and chloroplast and mitochondrial investment in the mestome sheath (MS), where CO2 is concentrated in C2 and C4 Neurachne species; width of the MS cells; frequency of plasmodesmata in the MS cell walls adjoining the parenchymatous bundle sheath; and the proportion of leaf GLDP invested in the MS tissue. Less than 12% of the leaf GLDP was allocated to the MS of completely C3 Neurachninae species with C * values of 56-61 µmol mol-1, whereas two-thirds of leaf GLDP was in the MS of Neurachne lanigera, which exhibits a newly-identified, partial C2 phenotype with C * of 44 µmol mol-1 Increased investment of GLDP in MS tissue of the C2 species was attributed to more MS mitochondria and less GLDP in mesophyll mitochondria. These results are consistent with a model where C4 evolution in Neurachninae initially occurred via an increase in organelle and GLDP content in MS cells, which generated a sink for photorespired CO2 in MS tissues.