ELEVATED CARBON DIOXIDE DIFFERENTIALLY ALTERS THE PHOTOPHYSIOLOGY OF THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) AND EMILIANIA HUXLEYI (HAPTOPHYTA)1

Increasing anthropogenic carbon dioxide is causing changes to ocean chemistry, which will continue in a predictable manner. Dissolution of additional atmospheric carbon dioxide leads to increased concentrations of dissolved carbon dioxide and bicarbonate and decreased pH in ocean water. The concomitant effects on phytoplankton ecophysiology, leading potentially to changes in community structure, are now a focus of concern. Therefore, we grew the coccolithophore Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler and the diatom strains Thalassiosira pseudonana (Hust.) Hasle et Heimdal CCMP 1014 and T. pseudonana CCMP 1335 under low light in turbidostat photobioreactors bubbled with air containing 390 ppmv or 750 ppmv CO₂. Increased pCO₂ led to increased growth rates in all three strains. In addition, protein levels of RUBISCO increased in the coastal strains of both species, showing a larger capacity for CO₂ assimilation at 750 ppmv CO₂. With increased pCO₂, both T. pseudonana strains displayed an increased susceptibility to PSII photoinactivation and, to compensate, an augmented capacity for PSII repair. Consequently, the cost of maintaining PSII function for the diatoms increased at increased pCO₂. In E. huxleyi, PSII photoinactivation and the counter‐acting repair, while both intrinsically larger than in T. pseudonana, did not change between the current and high‐pCO₂ treatments. The content of the photosynthetic electron transport intermediary cytochrome b6/f complex increased significantly in the diatoms under elevated pCO₂, suggesting changes in electron transport function.     

Induction of Phase Variation Events in the Life Cycle of the Marine Coccolithophorid Emiliania huxleyi

Emiliania huxleyi is a unicellular marine alga that is considered to be the world's major producer of calcite. The life cycle of this alga is complex and is distinguished by its ability to synthesize exquisitely sculptured calcium carbonate cell coverings known as coccoliths. These structures have been targeted by materials scientists for applications relating to the chemistry of biomedical materials, robust membranes for high-temperature separation technology, lightweight ceramics, and semiconductor design. To date, however, the molecular and biochemical events controlling coccolith production have not been determined. In addition, little is known about the life cycle of E. huxleyi and the environmental and physiological signals triggering phase switching between the diploid and haploid life cycle stages. We have developed laboratory methods for inducing phase variation between the haploid (S-cell) and diploid (C-cell) life cycle stages of E. huxleyi. Plating E. huxleyi C cells on solid media was shown to induce phase switching from the C-cell to the S-cell life cycle stage, the latter of which has been maintained for over 2 years under these conditions. Pure cultures of S cells were obtained for the first time. Laboratory conditions for inducing phase switching from the haploid stage to the diploid stage were also established. Regeneration of the C-cell stage from pure cultures of S cells followed a predictable pattern involving formation of large aggregations of S cells and the subsequent production of cultures consisting predominantly of diploid C cells. These results demonstrate the ability to manipulate the life cycle of E. huxleyi under controlled laboratory conditions, providing us with powerful tools for the development of genetic techniques for analysis of coccolithogenesis and for investigating the complex life cycle of this important marine alga.     

Transcriptome analysis of functional differentiation between haploid and diploid cells of Emiliania huxleyi, a globally significant photosynthetic calcifying cell

Background  

Eukaryotes are classified as either haplontic, diplontic, or haplo-diplontic, depending on which ploidy levels undergo mitotic cell division in the life cycle. Emiliania huxleyi is one of the most abundant phytoplankton species in the ocean, playing an important role in global carbon fluxes, and represents haptophytes, an enigmatic group of unicellular organisms that diverged early in eukaryotic evolution. This species is haplo-diplontic. Little is known about the haploid cells, but they have been hypothesized to allow persistence of the species between the yearly blooms of diploid cells. We sequenced over 38,000 expressed sequence tags from haploid and diploid E. huxleyi normalized cDNA libraries to identify genes involved in important processes specific to each life phase (2N calcification or 1N motility), and to better understand the haploid phase of this prominent haplo-diplontic organism.     

Increased CO<Subscript>2</Subscript> and the effect of pH on growth and calcification of <Emphasis Type="Italic">Pleurochrysis carterae</Emphasis> and <Emphasis Type="Italic">Emiliania huxleyi</Emphasis> (Haptophyta) in semicontinuous cultures

The effects of changes in CO₂ and pH on biomass productivity and carbon uptake of Pleurochrysis carterae and Emiliania huxleyi in open raceway ponds and a plate photobioreactor were studied. The pH of P. carterae cultures increased during day and decreased at night, whereas the pH of E. huxleyi cultures showed no significant diurnal changes. P. carterae coccolith production occurs during the dark period, whereas in E. huxleyi, coccolith production is mainly during the day. Addition of CO₂ at constant pH (pH-stat) resulted in an increase in P. carterae biomass and coccolith productivity, while CO₂ addition lowered E. huxleyi biomass and coccolith production. Neither of these algae could grow at less than pH 7.5. Species-specific diurnal pH and pCO₂ variations could be indicative of significant differences in carbon uptake between these two species. While E. huxleyi has been suggested to be predominantly a bicarbonate user, our results indicate that P. carterae may be using CO₂ as the main C source for photosynthesis and calcification.     

Strong shift from HCO3 − to CO2 uptake in Emiliania huxleyi with acidification: new approach unravels acclimation versus short-term pH effects

Effects of ocean acidification on Emiliania huxleyi strain RCC 1216 (calcifying, diploid life-cycle stage) and RCC 1217 (non-calcifying, haploid life-cycle stage) were investigated by measuring growth, elemental composition, and production rates under different pCO₂ levels (380 and 950 μatm). In these differently acclimated cells, the photosynthetic carbon source was assessed by a ¹⁴C disequilibrium assay, conducted over a range of ecologically relevant pH values (7.9–8.7). In agreement with previous studies, we observed decreased calcification and stimulated biomass production in diploid cells under high pCO₂, but no CO₂-dependent changes in biomass production for haploid cells. In both life-cycle stages, the relative contributions of CO₂ and HCO₃ ^? uptake depended strongly on the assay pH. At pH values ≤ 8.1, cells preferentially used CO₂ (≥ 90 % CO₂), whereas at pH values ≥ 8.3, cells progressively increased the fraction of HCO₃ ^? uptake (~45 % CO₂ at pH 8.7 in diploid cells; ~55 % CO₂ at pH 8.5 in haploid cells). In contrast to the short-term effect of the assay pH, the pCO₂ acclimation history had no significant effect on the carbon uptake behavior. A numerical sensitivity study confirmed that the pH-modification in the ¹⁴C disequilibrium method yields reliable results, provided that model parameters (e.g., pH, temperature) are kept within typical measurement uncertainties. Our results demonstrate a high plasticity of E. huxleyi to rapidly adjust carbon acquisition to the external carbon supply and/or pH, and provide an explanation for the paradoxical observation of high CO₂ sensitivity despite the apparently high HCO₃ ^? usage seen in previous studies.     

Vertical and lateral variations in coccolithophore community structure across the subtropical frontal zone in the South Atlantic Ocean

In this study the coccolithophore compositions of 11 plankton depth stations along a N–S transect from the South Atlantic Subtropical Gyre to the Subantarctic Zone were examined qualitatively and quantitatively. The lateral and vertical distribution patterns of not only the most abundant taxa but also of the morphotypes of distinct species complexes, such as Calcidiscus leptoporus, Emiliania huxleyi, and Umbellosphaera tenuis were the focus. Geographic variation among morphotypes mirrors different ecological affinities of the members of a species complex. Multivariate statistics were used to infer the relationship between a set of known environmental data and species concentrations. The results of the detrended Canonical Correspondence Analysis (CCA) revealed the presence of distinct species assemblages. The Subtropical Gyre assemblage within the upper 50 m of the photic zone is mainly composed of Umbellosphaera irregularis, U. tenuis types III and IV, Discosphaera tubifera, Rhabdosphaera clavigera, S. pulchra and E. huxleyi var. corona, adapted to warm and oligotrophic conditions. In the deeper photic zone abundant Florisphaera profunda, Gephyrocapsa ericsonii and Oolithotus spp. are encountered, benefiting from higher nutrient concentrations in the vicinity of the nutricline. A well-defined Subtropical Frontal Zone (STFZ) association is clearly dominated by E. huxleyi types A and C throughout the upper 100 m of the water column. Secondary contributors in the upper photic zone are Syracosphaera spp. (mainly S. histrica, S. molischii), Michaelsarsia elegans, Ophiaster spp. and U. tenuis type II. This assemblage is associated with cooler, nutrient-rich waters. E. huxleyi type B is found deeper in the water column. Here it is accompanied by Algirosphaera robusta, G. muellerae, and S. anthos indicating a tolerance of lower light availability in environments with elevated productivity. C. leptoporus spp. leptoporus shows relatively high cell numbers in all sampled water levels throughout the STFZ. Interestingly, its coccoliths are often smaller 5 μm in lith diameter. The mean coccolithophore assemblages of a station were compared to the underlying surface sediment assemblages. For the most part, the distribution of the morphotypes is reflected in the sedimentary archive, thus proving their potential as paleoecological proxies.     

Response of nannoplankton to major changes in sea-surface temperature and movements of hydrological fronts over Site DSDP 594 (south Chatham Rise, southeastern New Zealand), during the last 130 kyr

A high-resolution history of paleoceanographic changes in the subpolar waters of the southern margin of the Subtropical Convergence Zone during the last 130 kyr, is present in foraminiferal assemblages of DSDP Site 594. The foraminifera indicate that sea-surface temperatures during the Last Interglacial Climax were warmer than today, and that between substage 5d through to the end of isotope stage 2, temperatures were mostly cooler than Holocene temperatures. The paleotemperatures suggest that (1) the Subtropical Convergence was located over the site during substage 5e, later moving further north, then moving southwards to near the site during the Holocene, and (2) the Polar Front was positioned over the Site during glacial stages 6, 4, 2 and possibly parts of stage 3. Several major events are indicated by the nannofloral assemblages during these large changes in sea-surface temperature and associated reorganization of ocean circulation. First, the time-progressive trends between E. huxleyi and medium to large Gephyrocapsa are unique to this site, with E. huxleyi dominating over medium Gephyrocapsa during stages 5c-a, middle part of stage 4 and after the middle point of stage 3. This unusual trend may (at least partly) be caused by the shift of the Polar Front across the site. Second, upwelling flora (E. huxleyi and small placoliths) increase in abundance during stages 1, 3 and 5, suggesting that upwelling or disturbance of water stratification took place during the interglacials. Thirdly, there are no significant differences between the distribution patterns of the various morphotypes of medium to large Gephyrocapsa, and the combined value of all medium Gephyrocapsa increases in abundance during glacials (stages 2 and 4 and the end of stage 6), similar to the abundance trends in benthic foraminifera. Finally, subordinate nannofossil taxa also show distinctive. climatic trends during the last glacial cycle: (1) Syracosphaera spp. are present in increased abundance during warmer extremes in climate (substages 5e, 5a, and stage 1); (2) Coccolithus pelagicus and Calcidiscus leptoporus dominate the subordinate nannofossil taxa, and their relative proportions seem to provide a useful paleoceanographic index, with C. pelagicus dominating when the Polar Front Zone is over the site (stages 6, 4 and 2), whilst C. leptoporus is relatively more abundant when the STC is positioned over the site (stages 1 and 5e). Increased abundance of C. pelagicus also can indicate intensified coastal upwelling.     

INORGANIC CARBON TRANSPORT IN RELATION TO CULTURE AGE AND INORGANIC CARBON CONCENTRATION IN A HIGH-CALCIFYING STRAIN OF EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)1

The relationships among inorganic carbon transport, bicarbonate availability, intracellular pH, and culture age were investigated in high-calcifying cultures of Emiliania huxleyi (Lohmann) Hay & Mohler. Measurement of inorganic carbon transport by the silicone-oil centrifugation technique demonstrated that gadolinium, a potential Ca²⁺ channel inhibitor, blocked intracellular inorganic carbon uptake and photosynthetic ¹⁴CO₂₊ fixation in exponential-phase cells. In stationary-phase cells, the intracellular inorganic carbon concentration was unaffected by gadolinium. Gadolinium was also used to investigate the link between bicarbonate and Ca²⁺ transport in high-calcifying cells of E. huxleyi. Bicarbonate availability had significant and rapid effects on pH_i in exponential- but not in stationary-phase cells. 4′, 4′-Diisothiocyanostilbene-2,2′-disulfonic acid did not block bicarbonate uptake from the external medium by exponential-phase cells. Inorganic carbon utilization by exponential- and stationary-phase cells of Emiliania huxleyi was investigated using a pH drift technique in a closed system. Light-dependent alkalization of the medium by stationary-phase cells resulted in a final pH of 10.1 and was inhibited by dextran-bound sulphonamide, an inhibitor of external carbonic anhydrase. Exponential-phase cells did not generate a pH drift. Overall, the results suggest that for high-calcifying cultures of E. huxleyi the predominant pathway of inorganic carbon utilization differs in exponential and stationary phase cells of the same culture.     

ELEVATED ATMOSPHERIC CARBON DIOXIDE INCREASES ORGANIC CARBON FIXATION BY EMILIANIA HUXLEYI (HAPTOPHYTA), UNDER NUTRIENT‐LIMITED HIGH‐LIGHT CONDITIONS1

Phytoplankton play a key role in determining the partitioning of CO₂ between the atmosphere and the ocean on seasonal, interannual, and millennial time scales. The magnitude of biological draw‐down of atmospheric CO₂ and C storage in the oceans is affected by concurrent changes in other environmental factors, like nutrient supply. Furthermore, variations in carbon‐to‐nitrogen (C:N) and carbon‐to‐phosphorus (C:P) assimilation ratios modify the oceanic CO₂ storage capacity. Here we show that increased atmospheric CO₂ concentration enhances CO₂ fixation into organic matter by a noncalcifying strain of Emiliania huxleyi (Lohmann) Hay & Mohler only under certain conditions, namely high light and nutrient limitation. Enhanced organic matter production was accompanied by marked deviations of the C:N:P ratio from the canonical stoichiometry of marine particulate matter of 106:16:1 (C:N:P) known as the Redfield ratio. Increased cell organic carbon content, C:N, and C:P were observed at high light when growth was either nitrogen or phosphorus limited. Elevated CO₂ led to further increases in the particulate C:N and C:P ratios. Enhanced CO₂ uptake by phytoplankton such as E. huxleyi, in response to elevated atmospheric CO₂, could increase carbon storage in the nitrogen‐limited regions of the oceans and thus act as a negative feedback on rising atmospheric CO₂ levels.     

One‐year experiment on the physiological response of the Mediterranean crustose coralline alga,Lithophyllum cabiochae,to elevated pCO2 and temperature

The response of respiration, photosynthesis, and calcification to elevated pCO₂ and temperature was investigated in isolation and in combination in the Mediterranean crustose coralline alga Lithophyllum cabiochae. Algae were maintained in aquaria during 1 year at near‐ambient conditions of irradiance, at ambient or elevated temperature (+3°C), and at ambient (ca. 400 μatm) or elevated pCO₂ (ca. 700 μatm). Respiration, photosynthesis, and net calcification showed a strong seasonal pattern following the seasonal variations of temperature and irradiance, with higher rates in summer than in winter. Respiration was unaffected by pCO₂ but showed a general trend of increase at elevated temperature at all seasons, except in summer under elevated pCO₂. Conversely, photosynthesis was strongly affected by pCO₂ with a decline under elevated pCO₂ in summer, autumn, and winter. In particular, photosynthetic efficiency was reduced under elevated pCO₂. Net calcification showed different responses depending on the season. In summer, net calcification increased with rising temperature under ambient pCO₂ but decreased with rising temperature under elevated pCO₂. Surprisingly, the highest rates in summer were found under elevated pCO₂ and ambient temperature. In autumn, winter, and spring, net calcification exhibited a positive or no response at elevated temperature but was unaffected by pCO₂. The rate of calcification of L. cabiochae was thus maintained or even enhanced under increased pCO₂. However, there is likely a trade‐off with other physiological processes. For example, photosynthesis declines in response to increased pCO₂ under ambient irradiance. The present study reports only on the physiological response of healthy specimens to ocean warming and acidification, however, these environmental changes may affect the vulnerability of coralline algae to other stresses such as pathogens and necroses that can cause major dissolution, which would have critical consequence for the sustainability of coralligenous habitats and the budgets of carbon and calcium carbonate in coastal Mediterranean ecosystems.     

Cryopreservation of the coccolithophore, <Emphasis Type="Italic">Emiliania huxleyi</Emphasis> (Haptophyta,Prymnesiophyceae)

We studied the cryopreservation of the most common coccolithophore, Emiliania huxleyi which is considered as one of the main global carbon cycle participants. Both stages of this complex life cycle species were submitted to gradual addition of three distinct cryoprotectants: dimethylsulfoxide (7.5% v/v), methanol (5% v/v) and proline (0.5 M). They were then control-rate cooled (−5 °C min⁻¹) to −50 °C before plunging into liquid nitrogen. Free radical oxygen species have been proposed to occur in cells subjected to pre-freezing manipulation or to cooling. Therefore, catalase (preventing accumulation of hydroxyl radicals) was evaluated for its ability to improve cell viability before and after freezing-thawing challenge. With the exception of proline which induced a decrease in diploid cell proliferation, cryoprotectants had no deleterious effects. On the contrary, growth of the haploid stage was enhanced by each CPA treatment, suggesting mixotrophic growth. Cryopreservation succeeded when dimethylsulfoxide was used, and the late exponential phase was obtained as soon as the 15th post-thawing day. Cell densities were then similar to the unfrozen controls. Catalase had no beneficial effect on the ability of cells to grow, neither prior freezing nor after thawing. In comparison with former attempts to cryopreserve E. huxleyi in other culture collection centers, our protocols allowed faster recovery.     

Have we been underestimating the effects of ocean acidification in zooplankton?

Understanding how copepods may respond to ocean acidification (OA) is critical for risk assessments of ocean ecology and biogeochemistry. The perception that copepods are insensitive to OA is largely based on experiments with adult females. Their apparent resilience to increased carbon dioxide (pCO₂) concentrations has supported the view that copepods are ‘winners’ under OA. Here, we show that this conclusion is not robust, that sensitivity across different life stages is significantly misrepresented by studies solely using adult females. Stage‐specific responses to pCO₂ (385–6000 μatm) were studied across different life stages of a calanoid copepod, monitoring for lethal and sublethal responses. Mortality rates varied significantly across the different life stages, with nauplii showing the highest lethal effects; nauplii mortality rates increased threefold when pCO₂ concentrations reached 1000 μatm (year 2100 scenario) with LC₅₀ at 1084 μatm pCO₂. In comparison, eggs, early copepodite stages, and adult males and females were not affected lethally until pCO₂ concentrations ≥3000 μatm. Adverse effects on reproduction were found, with >35% decline in nauplii recruitment at 1000 μatm pCO₂. This suppression of reproductive scope, coupled with the decreased survival of early stage progeny at this pCO₂ concentration, has clear potential to damage population growth dynamics in this species. The disparity in responses seen across the different developmental stages emphasizes the need for a holistic life‐cycle approach to make species‐level projections to climate change. Significant misrepresentation and error propagation can develop from studies which attempt to project outcomes to future OA conditions solely based on single life history stage exposures.     

Regulation of CaCO(3) formation in coccolithophores

Coccolithophores impact the ocean carbon cycle principally through the generation of CO₂ during CaCO₃ production. Coccolithophore biomineralization has been examined most extensively in Pleurochrysis carterae and Emiliania huxleyi both of which produce mineralized scales—coccoliths—composed of elaborate calcite crystals attached to an underlying organic base plate. Calcification of preformed base plates is mediated by acidic polysaccharides and occurs in Golgi-derived structures known as mineralizing vesicles. In Pleurochrysis a high capacity calcium-binding polysaccharide PS2 is required for efficient nucleation of calcitic protocrystals. A galacturonomannan PS3 is required for the growth and transformation of the protocrystals into a massive double disc of calcite. The genes that regulate expression of the glycans have not yet been identified. In addition to the coccolith-bearing diploid phases, Pleurochrysis and Emiliania possess both haploid and diploid non-calcifying stages, which are self-perpetuating via binary fission. One non-calcifying Pleurochrysis phase fails to synthesis PS2 and spontaneously reverts to the mineralizing morphotype in laboratory cultures. As yet, there is little information on environmental factors that effect the expression or silencing of calcifying genes or favor the growth of calcifying over non-calcifying phases. These issues will need extensive investigation, if we are to appreciate the role of coccolithophores in the regulation of atmospheric CO₂ levels.     

Ocean acidification stimulates particulate organic carbon accumulation in two Antarctic diatom species under moderate and high natural solar radiation

Impacts of rising atmospheric CO₂ concentrations and increased daily irradiances from enhanced surface water stratification on phytoplankton physiology in the coastal Southern Ocean remain still unclear. Therefore, in the two Antarctic diatoms Fragilariopsis curta and Odontella weissflogii, the effects of moderate and high natural solar radiation combined with either ambient or future pCO₂ on cellular particulate organic carbon (POC) contents and photophysiology were investigated. Results showed that increasing CO₂ concentrations had greater impacts on diatom physiology than exposure to increasing solar radiation. Irrespective of the applied solar radiation regime, cellular POC quotas increased with future pCO₂ in both diatoms. Lowered maximum quantum yields of photochemistry in PSII (F_v/F_m) indicated a higher photosensitivity under these conditions, being counteracted by increased cellular concentrations of functional photosynthetic reaction centers. Overall, our results suggest that both bloom‐forming Antarctic coastal diatoms might increase carbon contents under future pCO₂ conditions despite reduced physiological fitness. This indicates a higher potential for primary productivity by the two diatom species with important implications for the CO₂ sequestration potential of diatom communities in the future coastal Southern Ocean.     

Long-term responsiveness to free air CO2 enrichment of functional types, species and genotypes of plants from fertile permanent grassland

To test inter- and intraspecific variability in the responsiveness to elevated CO₂, 9–14 different genotypes of each of 12 perennial species from fertile permanent grassland were grown in Lolium perenne swards under ambient (35 Pa) and elevated (60 Pa) atmospheric partial pressure of CO₂ (pCO₂) for 3 years in a free air carbon dioxide enrichment (FACE) experiment. The plant species were grouped according to their functional types: grasses (L. perenne, L. multiflorum, Arrhenatherum elatius, Dactylis glomerata, Festuca pratensis, Holcus lanatus, Trisetum flavescens), non-legume dicots (Rumex obtusifolius, R. acetosa, Ranunculus friesianus), and legumes (Trifolium repens, T. pratense). Yield (above a cutting height of 4.5 cm) was measured three times per year. The results were as follow. (1) There were highly significant differences in the responsiveness to elevated pCO₂ between the three functional types; legumes showed the strongest and grasses the weakest yield increase at elevated pCO₂. (2) There were differences in the temporal development of responsiveness to elevated pCO₂ among the functional types. The responsiveness of the legumes declined from the first to the second year, while the responsiveness of the non-legume dicots increased over the 3 years. During the growing season, the grasses and the non-legume dicots showed the strongest response to elevated pCO₂ during reproductive growth in the spring. (3) There were no significant genotypic differences in responsiveness to elevated pCO₂. Our results suggest that, due to interspecific differences in the responsiveness to elevated pCO₂, the species proportion within fertile temperate grassland may change if the increase in pCO₂ continues. Due to the temporal differences in the responsiveness to elevated pCO₂ among species, complex effects of elevated pCO₂ on competitive interactions in mixed swards must be expected. The existence of genotypic variability in the responsiveness to elevated pCO₂, on which selection could act, was not found under our experimental conditions. Received: 11 May 1997 / Accepted: 11 August 1997     

Effect of elevated atmospheric CO2 concentration on soil and root respiration in winter wheat by using a respiration partitioning chamber

Soil respiration in a cropland is the sum of heterotrophic (mainly microorganisms) and autotrophic (root) respiration. The contribution of both these types to soil respiration needs to be understood to evaluate the effects of environmental change on soil carbon cycling and sequestration. In this paper, the effects of free-air CO₂ enrichment (FACE) on hetero- and autotrophic respiration in a wheat field were differentiated and evaluated by a novel split-root growth and gas collection system. Elevated atmospheric pCO₂ of approximately 200 μmol mol⁻¹ above the ambient pCO₂ significantly increased soil respiration by 15.1 and 14.8% at high nitrogen (HN) and low nitrogen (LN) application rates, respectively. The effect of elevated atmospheric pCO₂ on root respiration was not consistent across the wheat growth stages. Elevated pCO₂ significantly increased and decreased root respiration at the booting-heading stage (middle stage) and the late-filling stage (late stage), respectively, in HN and LN treatments; however, no significant effect was found at the jointing stage (early stage). Thus, the effect of increased pCO₂ on cumulative root respiration for the entire wheat growing season was not significant. Cumulative root respiration accounted for approximately 25–30% of cumulative soil respiration in the entire wheat growing season. Consequently, cumulative microbial respiration (soil respiration minus root respiration) increased by 22.5 and 21.1% due to elevated pCO₂ in HN and LN, respectively. High nitrogen application significantly increased root respiration at the late stage under both elevated pCO₂ and ambient pCO₂; however, no significant effects were found on cumulative soil respiration, root respiration, and microbial respiration. These findings suggest that heterotrophic respiration, which is influenced by increased substrate supplies from the plant to the soil, is the key process to determine C emission from agro-ecosystems with regard to future scenarios of enriched pCO₂.