The role of photorespiration in preventing feedback regulation via ATP synthase in Nicotiana tabacum.

Photorespiration consumes substantial amounts of energy in the forms of adenosine triphosphate (ATP) and reductant making the pathway an important component in leaf energetics. Because of this high reductant demand, photorespiration is proposed to act as a photoprotective electron sink. However, photorespiration consumes more ATP relative to reductant than the C3 cycle meaning increased flux disproportionally increases ATP demand relative to reductant. Here we explore how energetic consumption from photorespiration impacts the flexibility of the light reactions in nicotiana tabacum. Specifically, we demonstrate that decreased photosynthetic efficiency (ϕII ) at low photorespiratory flux was related to feedback regulation at the chloroplast ATP synthase. Additionally, decreased ϕII at high photorespiratory flux resulted in the accumulation of photoinhibition at photosystem II centers. These results are contrary to the proposed role of photorespiration as a photoprotective electron sink. Instead, our results suggest a novel role of ATP consumption from photorespiration in maintaining ATP synthase activity, with implications for maintaining energy balance and preventing photodamage that will be critical for plant engineering strategies.

Stomatal dynamics in Alloteropsis semialata arise from the evolving interplay between photosynthetic physiology, stomatal size and biochemistry.

C4 plants are expected to have faster stomatal movements than C3 species because they tend to have smaller guard cells. However, little is known about how the evolution of C4 photosynthesis influences stomatal dynamics in relation to guard cell size and environmental factors. We studied photosynthetically diverse populations of the grass Alloteropsis semialata, showing that the origin of C4 photosynthesis in this species was associated with a shortening of stomatal guard and subsidiary cells. However, for a given cell size, C4 and C3-C4 intermediate individuals had similar or slower light-induced stomatal opening speeds than C3 individuals. Conversely, when exposed to decreasing light, stomata in C4 plants closed as fast as those in non-C4 plants. Polyploid formation in some C4 plants led to larger stomatal cells and was associated with slower stomatal opening. Conversely, diversification of C4 diploid plants into wetter environments was associated with an acceleration of stomatal opening. Overall, there was significant relationship between light-saturated photosynthesis and stomatal opening speed in the C4 plants, implying that photosynthetic energy production was limiting for stomatal opening. Stomatal dynamics in this wild grass therefore arise from the evolving interplay between photosynthetic physiology and the size and biochemical function of stomatal complexes.

Absence of carbonic anhydrase in chloroplasts affects C3 plant development but not photosynthesis.

The enzyme carbonic anhydrase (CA), which catalyzes the interconversion of bicarbonate with carbon dioxide (CO2) and water, has been hypothesized to play a role in C3 photosynthesis. We identified two tobacco stromal CAs, ß-CA1 and ß-CA5, and produced CRISPR/Cas9 mutants affecting their encoding genes. While single knockout lines Δß-ca1 and Δß-ca5 had no striking phenotypic differences compared to wild type (WT) plants, Δß-ca1ca5 leaves developed abnormally and exhibited large necrotic lesions even when supplied with sucrose. Leaf development of Δß-ca1ca5 plants normalized at 9,000 ppm CO2 Leaves of Δß-ca1ca5 mutants and WT that had matured in high CO2 had identical CO2 fixation rates and photosystem II efficiency. Fatty acids, which are formed through reactions with bicarbonate substrates, exhibited abnormal profiles in the chloroplast CA-less mutant. Emerging Δß-ca1ca5 leaves produce reactive oxygen species in chloroplasts, perhaps due to lower nonphotochemical quenching efficiency compared to WT. Δß-ca1ca5 seedling germination and development is negatively affected at ambient CO2 Transgenes expressing full-length ß-CA1 and ß-CA5 proteins complemented the Δß-ca1ca5 mutation but inactivated (ΔZn-ßCA1) and cytoplasm-localized (Δ62-ßCA1) forms of ß-CA1 did not reverse the growth phenotype. Nevertheless, expression of the inactivated ΔZn-ßCA1 protein was able to restore the hypersensitive response to tobacco mosaic virus, while Δß-ca1 and Δß-ca1ca5 plants failed to show a hypersensitive response. We conclude that stromal CA plays a role in plant development, likely through providing bicarbonate for biosynthetic reactions, but stromal CA is not needed for maximal rates of photosynthesis in the C3 plant tobacco.

Overexpression of chloroplastic Zea mays NADP-malic enzyme (ZmNADP-ME) confers tolerance to salt stress in Arabidopsis thaliana.

The C4 plants photosynthesize better than C3 plants especially in arid environment. As an attempt to genetically convert C3 plant to C4, the cDNA of decarboxylating C4 type NADP-malic enzyme from Zea mays (ZmNADP-ME) that has lower Km for malate and NADP than its C3 isoforms, was overexpressed in Arabidopsis thaliana under the control of 35S promoter. Due to increased activity of NADP-ME in the transgenics the malate decarboxylation increased that resulted in loss of carbon skeletons needed for amino acid and protein synthesis. Consequently, amino acid and protein content of the transgenics declined. Therefore, the Chl content, photosynthetic efficiency (Fv/Fm), electron transport rate (ETR), the quantum yield of photosynthetic CO2 assimilation, rosette diameter, and biomass were lower in the transgenics. However, in salt stress (150 mM NaCl), the overexpressers had higher Chl, protein content, Fv/Fm, ETR, and biomass than the vector control. NADPH generated in the transgenics due to increased malate decarboxylation, contributed to augmented synthesis of proline, the osmoprotectant required to alleviate the reactive oxygen species-mediated membrane damage and oxidative stress. Consequently, the glutathione peroxidase activity increased and H2O2 content decreased in the salt-stressed transgenics. The reduced membrane lipid peroxidation and lower malondialdehyde production resulted in better preservation, of thylakoid integrity and membrane architecture in the transgenics under saline environment. Our results clearly demonstrate that overexpression of C4 chloroplastic ZmNADP-ME in the C3 Arabidopsis thaliana, although decrease their photosynthetic efficiency, protects the transgenics from salinity stress.

Maize (Zea mays L.) planted at higher density utilizes dynamic light more efficiently.

In nature, plants are exposed to a dynamic light environment. Fluctuations in light decreased the photosynthetic light utilization efficiency (PLUE) of leaves, and much more severely in C4 species than in C3 species. However, little is known about the plasticity of PLUE under dynamic light in C4 species. Present study focused on the influence of planting density to the photosynthesis under dynamic light in maize (Zea mays L.), a most important C4 crop. In addition, the molecular mechanism behind photosynthetic adaptation to planting density were also explored by quantitative proteomics analysis. Results revealed that as planting density increases, maize leaves receive less light that fluctuates more. The maize planted at high density (HD) improved the PLUE under dynamic light, especially in the middle and later growth stages. Quantitative proteomics analysis showed that the transfer of nitrogen from Rubisco to RuBP regeneration and C4 pathway related enzymes contributes to the photosynthetic adaptation to lower and more fluctuating light environment in HD maize. This study provides potential ways to further improve the light energy utilization efficiency of maize in HD.

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.

Overexpression of cytoplasmic C4 Flaveria bidentis carbonic anhydrase in C3 Arabidopsis thaliana increases amino acids, photosynthetic potential, and biomass.

An important method to improve photosynthesis in C3 crops, such as rice and wheat, is to transfer efficient C4 characters to them. Here, cytosolic carbonic anhydrase (CA: ßCA3) of the C4 Flaveria bidentis (Fb) was overexpressed under the control of 35 S promoter in Arabidopsis thaliana, a C3 plant, to enhance its photosynthetic efficiency. Overexpression of CA resulted in a better supply of the substrate HCO3- for the endogenous phosphoenolpyruvate carboxylase in the cytosol of the overexpressers, and increased its activity for generating malate that feeds into the tricarboxylic acid cycle. This provided additional carbon skeleton for increased synthesis of amino acids aspartate, asparagine, glutamate, and glutamine. Increased amino acids contributed to higher protein content in the transgenics. Furthermore, expression of FbßCA3 in Arabidopsis led to a better growth due to expression of several genes leading to higher chlorophyll content, electron transport, and photosynthetic carbon assimilation in the transformants. Enhanced CO2 assimilation resulted in increased sugar and starch content, and plant dry weight. In addition, transgenic plants had lower stomatal conductance, reduced transpiration rate, and higher water-use efficiency. These results, taken together, show that expression of C4 CA in the cytosol of a C3 plant can indeed improve its photosynthetic capacity with enhanced water-use efficiency.

CAM photosynthesis: the acid test.

There is currently considerable interest in the prospects for bioengineering crassulacean acid metabolism (CAM) photosynthesis - or key elements associated with it, such as increased water-use efficiency - into C3 plants. Resolving how CAM photosynthesis evolved from the ancestral C3 pathway could provide valuable insights into the targets for such bioengineering efforts. It has been proposed that the ability to accumulate organic acids at night may be common among C3 plants, and that the transition to CAM might simply require enhancement of pre-existing fluxes, without the need for changes in circadian or diurnal regulation. We show, in a survey encompassing 40 families of vascular plants, that nocturnal acidification is a feature entirely restricted to CAM species. Although many C3 species can synthesize malate during the light period, we argue that the switch to night-time malic acid accumulation requires a fundamental metabolic reprogramming that couples glycolytic breakdown of storage carbohydrate to the process of net dark CO2 fixation. This central element of the CAM pathway, even when expressed at a low level, represents a biochemical capability not seen in C3 plants, and so is better regarded as a discrete evolutionary innovation than as part of a metabolic continuum between C3 and CAM.

Salt stress induces Kranz anatomy and expression of C4 photosynthetic enzymes in the amphibious sedge Eleocharis vivipara.

Eleocharis vivipara Link is a unique amphibious leafless plant of the Cyperaceae. The terrestrial form develops culms with Kranz anatomy and C4-like traits, while the submerged form does culms with non-Kranz anatomy and C3 traits. The submerged form develops new culms with C4-like mode when exposed to air or exogenous abscisic acid. In this study, we investigated whether salt stress (0.05-0.3 M NaCl) has a similar effect. When the submerged form was grown for one month in solutions of 0.1 M NaCl and more, culm growth was strongly suppressed. However, these plants slowly developed new culms that had Kranz anatomy with chloroplast-abundant Kranz bundle sheath cells. Although the culms of the submerged form had only few stomata, culms grown in the NaCl solution had many stomata. The NaCl-grown culms also accumulated large amounts of C4 photosynthetic enzymes (phosphoenolpyruvate carboxylase and pyruvate Pi dikinase), and the cellular localization patterns of these enzymes and ribulose 1,5-bisphosphate carboxylase/oxygenase were similar to those in terrestrial culms. Accumulation of C4 enzymes increased in mature culms of the submerged form (with non-Kranz anatomy) when exposed to 0.2 M NaCl solution for one week. These results suggest that salt stress induces development of Kranz anatomy and expression of C4 photosynthetic enzymes in the submerged C3 form of E. vivipara, whereas the anatomical and biochemical traits of C4 photosynthesis appear to be regulated independently.

Distinguishing carbon gains from photosynthesis and heterotrophy in C3-hemiparasite-C3-host pairs.

BACKGROUND AND AIMS: Previous carbon stable isotope (13C) analyses have shown for very few C3-hemiparasites utilizing C4- or CAM-hosts the use of two carbon sources, autotrophy and heterotrophy. This 13C approach, however, failed for the frequently occurring C3-C3 parasite-host pairs. Thus, we used hydrogen stable isotope (2H) natural abundances as a substitute for 13C within a C3-Orobanchaceae sequence graded by haustoria complexity and C3-Santalaceae. METHODS: Parasitic plants and their real or potential host plants as references were collected in Central European lowland and alpine mountain meadows and forests. Parasitic plants included the xylem-feeding holoparasite Lathraea squamaria parasitizing on the same carbon nutrient source (xylem-transported organic carbon compounds) as potentially Pedicularis, Rhinanthus, Bartsia, Melampyrum and Euphrasia hemiparasites. Reference plants were used for an autotrophy-only isotope baseline. A multi-element stable isotope natural abundance approach was applied. KEY RESULTS: Species-specific heterotrophic carbon gain ranging from 0 to 51 % was estimated by a 2H mixing-model. The sequence in heterotrophic carbon gain mostly met the morphological grading by haustoria complexity: Melampyrum- < Rhinanthus- < Pedicularis-type. CONCLUSION: Due to higher transpiration and lower water-use efficiency, depletion in 13C, 18O and 2H compared to C3-host plants should be expected for tissues of C3-hemiparasites. However, 2H is counterbalanced by transpiration (2H-depletion) and heterotrophy (2H-enrichment). Progressive 2H-enrichment can be used as a proxy to evaluate carbon gains from hosts.

Gibberellic acid induces non-Kranz anatomy with C4-like biochemical traits in the amphibious sedge Eleocharis vivipara.

MAIN CONCLUSION: Gibberellic acid induces photosynthetic tissues with non-Kranz anatomy and C4-like biochemical traits in terrestrial-form plants of Eleocharis vivipara. This suggests that the structural and biochemical traits are independently regulated. The amphibious leafless sedge, Eleocharis vivipara Link, develops culms (photosynthetic organs) with C4-like traits and Kranz anatomy under terrestrial conditions, and C3 traits and non-Kranz anatomy under submerged conditions. The conversion from C3 mode to C4-like mode in E. vivipara is reportedly mediated by abscisic acid. Here, we investigated the effects of gibberellic acid (GA) on the differentiation of anatomical and photosynthetic traits because GA is involved in heterophylly in aquatic plants. When 100 µM GA was sprayed on terrestrial plants, the newly developed culms had non-Kranz anatomy in the basal part and Kranz-like anatomy in the upper part. In the basal part, the mesophyll cells were well developed, whereas the Kranz (bundle sheath) cells were reduced and contained few chloroplasts and mitochondria. Stomatal frequency was lower in the basal part than in the upper part. Nevertheless, these tissues had abundant accumulation and high activities of C4 photosynthetic enzymes and had C4-like δ13C values, as seen in the culms of the terrestrial form. When submerged plants were grown under water containing GA-biosynthesis inhibitors (uniconazole or paclobutrazol), the new culms had Kranz anatomy. The culms developed under paclobutrazol had the C3 pattern of cellular accumulation of photosynthetic enzymes. These data suggest that GA induces production of photosynthetic tissues with non-Kranz anatomy in terrestrial plants of E. vivipara, without concomitant expression of C3 biochemical traits. The data also suggest that the differentiation of C4 structural and biochemical traits is regulated independently.

Differences in leaf anatomy determines temperature response of leaf hydraulic and mesophyll CO2 conductance in phylogenetically related C4 and C3 grass species.

Leaf hydraulic and mesophyll CO2 conductance are both influenced by leaf anatomical traits, however it is poorly understood how the temperature response of these conductances differs between C4 and C3 species with distinct leaf anatomy. This study investigated the temperature response of leaf hydraulic conductance (Kleaf ), stomatal (gs ) and mesophyll (gm ) conductance to CO2 , and leaf anatomical traits in phylogenetically related Panicum antidotale (C4 ) and P. bisulcatum (C3 ) grasses. The C4 species had lower hydraulic conductance outside xylem (Kox ) and Kleaf compared with the C3 species. However, the C4 species had higher gm compared with the C3 species. Traits associated with leaf water movement, Kleaf and Kox , increased with temperature more in the C3 than in the C4 species, whereas traits related to carbon uptake, Anet and gm , increased more with temperature in the C4 than the C3 species. Our findings demonstrate that, in addition to a CO2 concentrating mechanism, outside-xylem leaf anatomy in the C4 species P. antidotale favours lower water movement through the leaf and stomata that provides an additional advantage for greater leaf carbon uptake relative to water loss with increasing leaf temperature than in the C3 species P. bisulcatum.

Using a reaction-diffusion model to estimate day respiration and reassimilation of (photo)respired CO2 in leaves.

Methods using gas exchange measurements to estimate respiration in the light (day respiration Rd ) make implicit assumptions about reassimilation of (photo)respired CO2 ; however, this reassimilation depends on the positions of mitochondria. We used a reaction-diffusion model without making these assumptions to analyse datasets on gas exchange, chlorophyll fluorescence and anatomy for tomato leaves. We investigated how Rd values obtained by the Kok and the Yin methods are affected by these assumptions and how those by the Laisk method are affected by the positions of mitochondria. The Kok method always underestimated Rd . Estimates of Rd by the Yin method and by the reaction-diffusion model agreed only for nonphotorespiratory conditions. Both the Yin and Kok methods ignore reassimilation of (photo)respired CO2 , and thus underestimated Rd for photorespiratory conditions, but this was less so in the Yin than in the Kok method. Estimates by the Laisk method were affected by assumed positions of mitochondria. It did not work if mitochondria were in the cytosol between the plasmamembrane and the chloroplast envelope. However, mitochondria were found to be most likely between the tonoplast and chloroplasts. Our reaction-diffusion model effectively estimates Rd , enlightens the dependence of Rd estimates on reassimilation and clarifies (dis)advantages of existing methods.

Effects of drought and fire on resprouting capacity of 52 temperate Australian perennial native grasses.

It remains uncertain how perennial grasses with different photosynthetic pathways respond to fire, and how this response varies with stress at the time of burning. Resprouting after fire was examined in relation to experimentally manipulated pre-fire watering frequencies. We asked the following questions: are there response differences to fire between C3 and C4 grasses? And, how does post-fire resprouting vary with pre-fire drought stress? Fifty-two perennial Australian grasses (37 genera, 13 tribes) were studied. Three watering frequencies were applied to simulate increasing drought. Pre-fire tiller number, tiller density, specific leaf area and leaf dry matter content were measured as explanatory variables to assess response. Most species (90%) and individuals (79%) resprouted following experimental burning. C4 grasses had higher probabilities of surviving fire relative to C3 grasses. Responses were not related to phylogeny or tribe. High leaf dry matter content reduced the probability of dying, but also reduced the re-emergence of tillers. Post-fire tiller number increased with increasing drought, regardless of photosynthetic type, suggesting that drought plays a role in the ability of grasses to recover after fire. This has implications for understanding the persistence of species in landscapes where fire management is practiced.

Uncertainties and limitations of using carbon-13 and oxygen-18 leaf isotope exchange to estimate the temperature response of mesophyll CO2 conductance in C3 plants.

The internal CO2 gradient imposed by mesophyll conductance (gm ) reduces substrate availability for C3 photosynthesis. With several assumptions, estimates of gm can be made from coupled leaf gas exchange with isoflux analysis of carbon ∆13 C-gm and oxygen in CO2 , coupled with transpired water (H2 O) ∆18 O-gm to partition gm into its biochemical and anatomical components. However, these assumptions require validation under changing leaf temperatures. To test these assumptions, we measured and modeled the temperature response (15-40°C) of ∆13 C-gm and ∆18 O-gm along with leaf biochemistry in the C3 grass Panicum bisulcatum, which has naturally low carbonic anhydrase activity. Our study suggests that assumptions regarding the extent of isotopic equilibrium (θ) between CO2 and H2 O at the site of exchange, and that the isotopic composition of the H2 O at the sites of evaporation ( δw-e18 ) and at the site of exchange ( δw-ce18 ) are similar, may lead to errors in estimating the ∆18 O-gm temperature response. The input parameters for ∆13 C-gm appear to be less sensitive to temperature. However, this needs to be tested in species with diverse carbonic anhydrase activity. Additional information on the temperature dependency of cytosolic and chloroplastic pH may clarify uncertainties used for ∆18 O-gm under changing leaf temperatures.

No evidence for triose phosphate limitation of light-saturated leaf photosynthesis under current atmospheric CO2 concentration.

The triose phosphate utilization (TPU) rate has been identified as one of the processes that can limit terrestrial plant photosynthesis. However, we lack a robust quantitative assessment of TPU limitation of photosynthesis at the global scale. As a result, TPU, and its potential limitation of photosynthesis, is poorly represented in terrestrial biosphere models (TBMs). In this study, we utilized a global data set of photosynthetic CO2 response curves representing 141 species from tropical rainforests to Arctic tundra. We quantified TPU by fitting the standard biochemical model of C3 photosynthesis to measured photosynthetic CO2 response curves and characterized its instantaneous temperature response. Our results demonstrate that TPU does not limit leaf photosynthesis at the current ambient atmospheric CO2 concentration. Furthermore, our results showed that the light-saturated photosynthetic rates of plants growing in cold environments are not more often limited by TPU than those of plants growing in warmer environments. In addition, our study showed that the instantaneous temperature response of TPU is distinct from temperature response of the maximum rate of Rubisco carboxylation. The new formulations of the temperature response of TPU derived in this study may prove useful in quantifying the biochemical limits to terrestrial plant photosynthesis and improve the representation of plant photosynthesis in TBMs.

Reduced plant water status under sub-ambient pCO2 limits plant productivity in the wild progenitors of C3 and C4 cereals.

BACKGROUND AND AIMS: The reduction of plant productivity by low atmospheric CO2 partial pressure (pCO2) during the last glacial period is proposed as a limiting factor for the establishment of agriculture. Supporting this hypothesis, previous work has shown that glacial pCO2 limits biomass in the wild progenitors of C3 and C4 founder crops, in part due to the direct effects of glacial pCO2 on photosynthesis. Here, we investigate the indirect role of pCO2 mediated via water status, hypothesizing that faster soil water depletion at glacial (18 Pa) compared to post-glacial (27 Pa) pCO2, due to greater stomatal conductance, feeds back to limit photosynthesis during drying cycles. METHODS: We grew four wild progenitors of C3 and C4 crops at glacial and post-glacial pCO2 and investigated physiological changes in gas exchange, canopy transpiration, soil water content and water potential between regular watering events. Growth parameters including leaf area were measured. KEY RESULTS: Initial transpiration rates were higher at glacial pCO2 due to greater stomatal conductance. However, stomatal conductance declined more rapidly over the soil drying cycle in glacial pCO2 and was associated with decreased intercellular pCO2 and lower photosynthesis. Soil water content was similar between pCO2 levels as larger leaf areas at post-glacial pCO2 offset the slower depletion of water. Instead the feedback could be linked to reduced plant water status. Particularly in the C4 plants, soil-leaf water potential gradients were greater at 18 Pa compared with 27 Pa pCO2, suggesting an increased ratio of leaf evaporative demand to supply. CONCLUSIONS: Reduced plant water status appeared to cause a negative feedback on stomatal aperture in plants at glacial pCO2, thereby reducing photosynthesis. The effects were stronger in C4 species, providing a mechanism for reduced biomass at 18 Pa. These results have added significance when set against the drier climate of the glacial period.

Effects of elevated CO2 on photosynthetic traits of native and invasive C3 and C4 grasses.

BACKGROUND: Rising CO2 is expected to result in changes in plant traits that will increase plant productivity for some functional groups. Differential plant responses to elevated CO2 are likely to drive changes in competitive outcomes, with consequences for community structure and plant diversity. Many of the traits that are enhanced under elevated CO2 also confer competitive success to invasive species, and it is widely believed that invasive species will be more successful in high CO2. However, this is likely to depend on plant functional group, and evidence suggests that C3 plants tend to respond more strongly to CO2. RESULTS: We tested the hypothesis that invasive species would be more productive than noninvasive species under elevated CO2 and that stronger responses would be seen in C3 than C4 plants. We examined responses of 15 grass species (eight C3, seven C4), classified as noninvasive or invasive, to three levels of CO2 (390, 700 and 1000 ppm) in a closed chamber experiment. Elevated CO2 decreased conductance and %N and increased shoot biomass and C/N ratio across all species. Differences between invasive and noninvasive species depended on photosynthetic mechanism, with more differences for traits of C3 than C4 plants. Differences in trait means between invasive and noninvasive species tended to be similar across CO2 levels for many of the measured responses. However, noninvasive C3 grasses were more responsive than invasive C3 grasses in increasing tiller number and root biomass with elevated CO2, whereas noninvasive C4 grasses were more responsive than invasive C4 grasses in increasing shoot and root biomass with elevated CO2. For C3 grasses, these differences could be disadvantageous for noninvasive species under light competition, whereas for C4 grasses, noninvasive species may become better competitors with invasive species under increasing CO2. CONCLUSIONS: The ecophysiological mechanisms underlying invasion success of C3 and C4 grasses may differ. However, given that the direction of trait differences between invasive and noninvasive grasses remained consistent under ambient and elevated CO2, our results provide evidence that increases in CO2 are unlikely to change dramatically the competitive hierarchy of grasses in these functional groups.