Ecotrons: Powerful and versatile ecosystem analysers for ecology, agronomy and environmental science.

Ecosystems integrity and services are threatened by anthropogenic global changes. Mitigating and adapting to these changes require knowledge of ecosystem functioning in the expected novel environments, informed in large part through experimentation and modelling. This paper describes 13 advanced controlled environment facilities for experimental ecosystem studies, herein termed ecotrons, open to the international community. Ecotrons enable simulation of a wide range of natural environmental conditions in replicated and independent experimental units while measuring various ecosystem processes. This capacity to realistically control ecosystem environments is used to emulate a variety of climatic scenarios and soil conditions, in natural sunlight or through broad-spectrum lighting. The use of large ecosystem samples, intact or reconstructed, minimizes border effects and increases biological and physical complexity. Measurements of concentrations of greenhouse trace gases as well as their net exchange between the ecosystem and the atmosphere are performed in most ecotrons, often quasi continuously. The flow of matter is often tracked with the use of stable isotope tracers of carbon and other elements. Equipment is available for measurements of soil water status as well as root and canopy growth. The experiments ran so far emphasize the diversity of the hosted research. Half of them concern global changes, often with a manipulation of more than one driver. About a quarter deal with the impact of biodiversity loss on ecosystem functioning and one quarter with ecosystem or plant physiology. We discuss how the methodology for environmental simulation and process measurements, especially in soil, can be improved and stress the need to establish stronger links with modelling in future projects. These developments will enable further improvements in mechanistic understanding and predictive capacity of ecotron research which will play, in complementarity with field experimentation and monitoring, a crucial role in exploring the ecosystem consequences of environmental changes.

Direct evidence for modulation of photosynthesis by an arbuscular mycorrhiza-induced carbon sink strength.

It has been suggested that plant carbon (C) use by symbiotic arbuscular mycorrhizal fungi (AMF) may be compensated by higher photosynthetic rates because fungal metabolism creates a strong C sink that prevents photosynthate accumulation and downregulation of photosynthesis. This mechanism remains largely unexplored and lacks experimental evidence. We report here two experiments showing that the experimental manipulation of the mycorrhizal C sink significantly affected the photosynthetic rates of cucumber host plants. We expected that a sudden reduction in sink strength would cause a significant reduction in photosynthetic rates, at least temporarily. Excision of part of the extraradical mycorrhizal mycelium from roots, and causing no disturbance to the plant, induced a sustained (10-40%) decline in photosynthetic rates that lasted from 30 min to several hours in plants that were well-nourished and hydrated, and in the absence of growth or photosynthesis promotion by mycorrhizal inoculation. This effect was though minor in plants growing at high (700 ppm) atmospheric CO2 . This is the first direct experimental evidence for the C sink strength effects exerted by arbuscular mycorrhizal symbionts on plant photosynthesis. It encourages further experimentation on mycorrhizal source-sink relations, and may have strong implications in large-scale assessments and modelling of plant photosynthesis.

Long-term and realistic global change manipulations had low impact on diversity of soil biota in temperate heathland.

In a dry heathland ecosystem we manipulated temperature (warming), precipitation (drought) and atmospheric concentration of CO2 in a full-factorial experiment in order to investigate changes in below-ground biodiversity as a result of future climate change. We investigated the responses in community diversity of nematodes, enchytraeids, collembolans and oribatid mites at two and eight years of manipulations. We used a structural equation modelling (SEM) approach analyzing the three manipulations, soil moisture and temperature, and seven soil biological and chemical variables. The analysis revealed a persistent and positive effect of elevated CO2 on litter C:N ratio. After two years of treatment, the fungi to bacteria ratio was increased by warming, and the diversities within oribatid mites, collembolans and nematode groups were all affected by elevated CO2 mediated through increased litter C:N ratio. After eight years of treatment, however, the CO2-increased litter C:N ratio did not influence the diversity in any of the four fauna groups. The number of significant correlations between treatments, food source quality, and soil biota diversities was reduced from six to three after two and eight years, respectively. These results suggest a remarkable resilience within the soil biota against global climate change treatments in the long term.

A replicated climate change field experiment reveals rapid evolutionary response in an ecologically important soil invertebrate.

Whether species can respond evolutionarily to current climate change is crucial for the persistence of many species. Yet, very few studies have examined genetic responses to climate change in manipulated experiments carried out in natural field conditions. We examined the evolutionary response to climate change in a common annelid worm using a controlled replicated experiment where climatic conditions were manipulated in a natural setting. Analyzing the transcribed genome of 15 local populations, we found that about 12% of the genetic polymorphisms exhibit differences in allele frequencies associated to changes in soil temperature and soil moisture. This shows an evolutionary response to realistic climate change happening over short-time scale, and calls for incorporating evolution into models predicting future response of species to climate change. It also shows that designed climate change experiments coupled with genome sequencing offer great potential to test for the occurrence (or lack) of an evolutionary response.

Grain protein concentration and harvestable protein under future climate conditions. A study of 108 spring barley accessions.

In the present study a set of 108 spring barley (H. vulgare L.) accessions were cultivated under predicted future levels of temperature and [CO2] as single factors and in combination (IPCC, AR5, RCP8.5). Across all genotypes, elevated [CO2] (700 ppm day/night) slightly decreased protein concentration by 5%, while elevated temperature (+5 °C day/night) substantially increased protein concentration by 29%. The combined treatment increased protein concentration across accessions by 8%. This was an increase less than predicted from strictly additive effects of the individual treatments. Despite the increase in grain protein concentration, the decrease in grain yield at combined elevated temperature and elevated [CO2] resulted in 23% less harvestable protein. There was variation in the response of the 108 accessions, which might be exploited to at least maintain if not increase harvestable grain protein under future climate change conditions.

Reduction of molecular gas diffusion through gaskets in leaf gas exchange cuvettes by leaf-mediated pores.

There is an ongoing debate on how to correct leaf gas exchange measurements for the unavoidable diffusion leakage that occurs when measurements are done in non-ambient CO2 concentrations. In this study, we present a theory on how the CO2 diffusion gradient over the gasket is affected by leaf-mediated pores (LMP) and how LMP reduce diffusive exchange across the gaskets. Recent discussions have so far neglected the processes in the quasi-laminar boundary layer around the gasket. Counter intuitively, LMP reduce the leakage through gaskets, which can be explained by assuming that the boundary layer at the exterior of the cuvette is enriched with air from the inside of the cuvette. The effect can thus be reduced by reducing the boundary layer thickness. The theory clarifies conflicting results from earlier studies. We developed leaf adaptor frames that eliminate LMP during measurements on delicate plant material such as grass leaves with circular cross section, and the effectiveness is shown with respiration measurements on a harp of Deschampsia flexuosa leaves. We conclude that the best solution for measurements with portable photosynthesis systems is to avoid LMP rather than trying to correct for the effects.

Solar UV-B effects on PSII performance in Betula nana are influenced by PAR level and reduced by EDU: results of a 3-year experiment in the High Arctic.

The long-term and diurnal responses of photosystem II (PSII) performance to near-ambient UV-B radiation were investigated in High Arctic Betula nana. We conducted an UV exclusion experiment with five replicated blocks consisting of open control (no filter), photosynthetic active radiation and UV-B transparent filter control (Teflon), UV-B-absorbing filter (Mylar) and UV-AB-absorbing filter (Lexan). Ethylenediurea (EDU), a chemical normally used to protect plants against ozone injury, was sprayed on the leaves both in the field and in an additional laboratory study to investigate if EDU mitigated the effects of UV-B. Chlorophyll-a fluorescence induction curves were used for analysis of OJIP test parameters. Near-ambient UV-B radiation reduced across season maximum quantum yield (TR(o) /ABS = F(v) /F(m)), approximated number of active PSII reaction center (RC/ABS) and the performance index (PI(ABS)), despite improved leaf screening against UV-B with higher content of UV-B-absorbing compounds and a lower specific leaf area. EDU application counteracted the negative impact of UV-B on TR(o) /ABS, RC/ABS and PI(ABS) . This indicates that the mechanisms behind UV-B and ozone damage share some common features. The midday depression was present in all treatments, but TR(o) /ABS and PI(ABS) were persistently lower in near-ambient UV-B compared to UV-B reduction. The recovery phase was particularly impaired in near-ambient UV-B and interactive effects between treatment × hour raised TR(o) /ABS, RC/ABS and PI(ABS) higher in reduced UV-B compared to near-ambient UV-B. This demonstrates current solar UV-B to reduce the PSII performance both on a daily as well as a seasonal basis in this High Arctic species.

Long-term structural canopy changes sustain net photosynthesis per ground area in high arctic Vaccinium uliginosum exposed to changes in near-ambient UV-B levels.

Full recovery of the ozone layer is not expected for several decades and consequently, the incoming level of solar ultraviolet-B (UV-B) will only slowly be reduced. Therefore to investigate the structural and photosynthetic responses to changes in solar UV-B we conducted a 5-year UV-B exclusion study in high arctic Greenland. During the growing season, the gas exchange (H2O and CO2) and chlorophyll-a fluorescence were measured in Vaccinium uliginosum. The leaf dry weight, carbon, nitrogen, stable carbon isotope ratio, chlorophyll and carotenoid content were determined from a late season harvest. The net photosynthesis per leaf area was on average 22% higher in 61% reduced UV-B treatment across the season, but per ground area photosynthesis was unchanged. The leaf level increase in photosynthesis was accompanied by increased leaf nitrogen, higher stomatal conductance and F(v)/F(m). There was no change in total leaf biomass, but reduction in total leaf area caused a pronounced reduction of specific leaf area and leaf area index in reduced UV-B. This demonstrates the structural changes to counterbalance the reduced plant carbon uptake seen per leaf area in ambient UV-B as the resulting plant carbon uptake per ground area was not affected. Thus, our understanding of long-term responses to UV-B reduction must take into account both leaf level processes as well as structural changes to understand the apparent robustness of plant carbon uptake per ground area. In this perspective, V. uliginosum seems able to adjust plant carbon uptake to the present amount of solar UV-B radiation in the High Arctic.

Terrestrial plant methane production and emission.

In this minireview, we evaluate all experimental work published on the phenomenon of aerobic methane (CH(4) ) generation in terrestrial plants and plant. Clearly, despite much uncertainty and skepticism, we conclude that the phenomenon is true. Four stimulating factors have been observed to induce aerobic plant CH(4) production, i.e. cutting injuries, increasing temperature, ultraviolet radiation and reactive oxygen species. Further, we analyze rates of measured emission of aerobically produced CH(4) in pectin and in plant tissues from different studies and argue that pectin is very far from the sole contributing precursor. In consequence, scaling up of aerobic CH(4) emission needs to take into consideration other potential sources than pectin. Due to the large uncertainties related to effects of stimulating factors, genotypic responses and type of precursors, we conclude that current attempts for upscaling aerobic CH(4) into a global budget is inadequate. Thus it is too early to draw the line under the aerobic methane emission in plants. Future work is needed for establishing the relative contribution of several proven potential CH(4) precursors in plant material.

Interactive effects of drought, elevated CO2 and warming on photosynthetic capacity and photosystem performance in temperate heath plants.

Increased temperature, atmospheric CO(2) and change in precipitation patterns affect plant physiological and ecosystem processes. In combination, the interactions between these effects result in complex responses that challenge our current understanding. In a multi-factorial field experiment with elevated CO(2) (CO2, FACE), nighttime warming (T) and periodic drought (D), we investigated photosynthetic capacity and PSII performance in the evergreen dwarf shrub Calluna vulgaris and the grass Deschampsia flexuosa in a temperate heath ecosystem. Photosynthetic capacity was evaluated using A/C(i) curves, leaf nitrogen content and chlorophyll-a fluorescence OJIP induction curves. The PSII performance was evaluated via the total performance index PI(total), which integrates the function of antenna, reaction centers, electron transport and end-acceptor reduction according to the OJIP-test. The PSII performance was negatively influenced by high air temperature, low soil water content and high irradiance dose. The experimental treatments of elevated CO(2) and prolonged drought generally down-regulated J(max), V(cmax) and PI(total). Recovery from these depressions was found in the evergreen shrub after rewetting, while post-rewetting up-regulation of these parameters was observed in the grass. Warming effects acted indirectly to improve early season J(max), V(cmax) and PI(total). The responses in the multi-factorial experimental manipulations demonstrated complex interactive effects of T×CO2, D×CO2 and T×D×CO2 on photosynthetic capacity and PSII performance. The impact on the O-J, J-I and I-P phases which determine the response of PI(total) are discussed. The single factor effects on PSII performance and their interactions could be explained by parallel adjustments of V(cmax), J(max) and leaf nitrogen in combination. Despite the highly variable natural environment, the OJIP-test was very robust in detecting the impacts of T, D, CO2 and their interactions. This study demonstrates that future climate will affect fundamental plant physiological processes in a way that is not predictable from single factor treatments. The interaction effects that were observed depended upon both the growth strategy of the species considered, and their ability to adjust during drought and rewetting periods.

Estimating daytime ecosystem respiration from eddy-flux data.

To understand what governs the patterns of net ecosystem exchange of CO2, an understanding of factors influencing the component fluxes, ecosystem respiration and gross primary production is needed. In the present paper, we introduce an alternative method for estimating daytime ecosystem respiration based on whole ecosystem fluxes from a linear regression of photosynthetic photon flux density data vs. daytime net ecosystem exchange data at forest ecosystem level. This method is based on the principles of the Kok-method applied at leaf level for estimating daytime respiration. We demonstrate the method with field data and provide a discussion of the limitations of the method.

Improved UV-B screening capacity does not prevent negative effects of ambient UV irradiance on PSII performance in High Arctic plants. Results from a six year UV exclusion study.

Long-term responses of ambient solar ultraviolet (UV) radiation were investigated on Salix arctica and Vaccinium uliginosum in a High Arctic heath ecosystem in Zackenberg, northeast Greenland. Over a period of six years, UV exclusion was conducted in the growing season by means of filters: 60% UV-B reduction, 90% UV-B+UV-A reduction, UV transparent filter control, and an open control without filter. Plant responses were evaluated using specific leaf area, leaf content of UV-B absorbing compounds and PSII performance parameters derived from chlorophyll-a fluorescence induction curves. Based on the JIP-test, we calculated the total performance index PI(total), which includes the integrating antennae, the PSII reaction center, intersystem electron transport and reduction of PSI end acceptors-dependent parameters. In both species, UV exclusion significantly decreased the content of UV-B-absorbing compounds. Salix increased its specific leaf area, while Vaccinium decreased it. UV exclusion increased the PI(total) in both species during all six years of experimentation. This response was governed by a significantly decreased RC/ABS, a marginally non-significant increased ET(o)/TR(o) and a significantly increased TR(o)/ABS=F(V)/F(M) and RE(o)/ET(o). These results demonstrate the current level of ambient UV-B to decrease PSII performance significantly in these High Arctic plants. It appears that the two plant species both have improved their UV-screening capacity, but through different strategies, although this did not sufficiently prevent negative effects of the ambient UV radiation. We argue the decreased PSII performance to be part of a response decreasing plant carbon uptake. We speculate the negative effects on PSII performance mediated by ambient UV irradiance to be present in years where warming induces early snowmelt, exposing the vegetation to high spring UV-B, and to be present in the future to the degree the ozone layer is not fully recovered.

Ambient UV-B radiation decreases photosynthesis in high arctic Vaccinium uliginosum.

An UV-B-exclusion experiment was established in high arctic Zackenberg, Northeast Greenland, to investigate the possible effects of ambient UV-B on plant performance. During almost a whole growing season, canopy gas exchange and Chl fluorescence were measured on Vaccinium uliginosum (bog blueberry). Leaf area, biomass, carbon, nitrogen and UV-B-absorbing compounds were determined from a late season harvest. Compared with the reduced UV-B treatment, the plants in ambient UV-B were found to have a higher content of UV-B-absorbing compounds, and canopy net photosynthesis was as an average 23% lower during the season. By means of the JIP-test, it was found that the potential of processing light energy through the photosynthetic machinery was slightly reduced in ambient UV-B. This indicates that not only the UV-B effects on PSII may be responsible for some of the observed reduction of photosynthesis but also the effects on other parts of the photosynthetic machinery, e.g. the Calvin cycle, might be important. The 60% reduction of the UV-B irradiance used in this study implies a higher relative change in the UV-B load than many of the supplemental experiments do, but the substantial effect on photosynthesis clearly indicates that V. uliginosum is negatively affected by the current level of UV-B.

Does the direct effect of atmospheric CO2 concentration on leaf respiration vary with temperature? Responses in two species of Plantago that differ in relative growth rate.

The objective of this study was to investigate the direct effect of elevated atmospheric CO2 concentrations on leaf respiration in darkness (R) over a broad range of measurement temperatures. Our aim was to further elucidate the underlying mechanism(s) of the often-reported inhibition of leaf R by a doubling of the atmospheric CO2 concentration. Experiments were conducted using two species of Plantago that differed in maximum relative growth rate (fast-growing Plantago lanceolata L. and the slow-growing P. euryphylla Briggs, Carolin & Pulley). Rates of leaf respiration (R) were measured at atmospheric CO2 concentrations ranging from 75 to 2000 &mgr;mol mol-1 at temperatures from 12 to 42 degrees C. R was measured as CO2 release with a portable gas exchange system with infrared gas analysers. Our hypothesis was that the changes in temperature alter the flux coefficient (i.e. the extent to which changes in potential enzyme activity has an effect on the rate of a reaction) of enzymes potentially affected by CO2. Initial analysis of our results suggested that R was inhibited by elevated CO2 in both species, with the apparent degree of inhibition being greatest at low temperature. Moreover, the apparent degree of inhibition following a doubling of atmospheric CO2 concentration from 350 to 700 &mgr;mol mol-1 was similar to that reported by several previous studies (approximately 14% and 26% for P. lanceolata and P. euryphylla, respectively) at a temperature equal to the mean of the previous studies. However, subsequent correction for diffusion leaks of CO2 across the gas exchange's cuvette gaskets revealed that no significant inhibition had occurred in either species, at any temperature. The inhibitory effect of elevated CO2 on leaf respiratory CO2 release reported by previous studies may therefore have been overestimated.