Cryopreservation of some useful microalgae species for biotechnological exploitation

Culture collections of microalgae represent a biological resource for scientific research and biotechnological applications. When compared to the current methods of maintenance and sub-culturing, cryopreservation minimizes labor costs and is an effective method for maintaining a large range of species over long periods with high stability. In order to determine the best cryopreservation method for microalgae species with great biotechnological potential, three freezing protocols were employed using different cryoprotectants (dimethyl sulfoxide—Me₂SO; methanol—MeOH). Three marine microalgae species (Thalassiosira weissflogii; Nannochloropsis oculata, and Skeletonema sp.) were cooled by directly plunging into liquid nitrogen (?196°C) and with two-step controlled cooling protocols (?18°C and ?80°C pre-treatments). After storage periods ranging from 10 to 120 days, viability was determined by the ability of cells to actively grow again. Results obtained for T. weissflogii showed that this species could be preserved at ultra-low temperature (?196°C) for 10 and 30 days with 10?% Me₂SO and 5?% MeOH when employed a controlled cooling protocol (?80°C). N. oculata was successfully cryopreserved either by direct freezing or with controlled cooling protocols. N. oculata samples presented good responses when treated with 5?% Me₂SO, 10?% Me₂SO, 5?% MeOH and even without any cryoprotectant. Skeletonema sp. did not survive cryopreservation in any of the tested conditions. The results indicate the difficulty in establishing common protocols for different microalgae species, being necessary further studies for a better understanding of cell damages during freezing and thawing conditions for each species.     

Stable carbon isotope ratio of tree leaves, boles and fine litter in a tropical forest in Rondônia, Brazil

Leaves of 208 trees were collected for isotopic analysis together with wood from 36 tree boles and 18 samples of fine litter from a terra-firme forest located at Samuel Ecological Reserve, Rondônia State, in the southwestern Amazon region. The range of δ¹³C values in leaves was from ?28 to ?36‰, with an average (±1 SD) of ?32.1?±?1.5‰, which was more negative than the δ¹³C values of bole samples (?28.4?±?2.0‰) and fine litter (?28.7?±?2.0‰). These values are within the range found for tropical and subtropical forests. Pooling the δ¹³C values for leaf samples from trees of the same height gave averages which were positively correlated with plant height at a highly significant level, with a slope of 0.06 and an intercept of ?33.3‰ and a correlation coefficient r ²=0.70 (P<0.001).     

Argentinean native wood species: Physical and mechanical characterization of some Prosopis species and Acacia aroma (Leguminosae; Mimosoideae)

One of the problems in marketing the wood of Prosopis and Acacia is the lack of standardization of its qualities. The aim of this paper was to obtain a preliminary detection of some properties of the wood of four species of the genus Prosopis and one species from Acacia grown in Argentina. To accomplish this objective, the content of extractives and some physical and mechanical characteristics were analyzed.The density ρ₁₂ of all the species indicates that these woods range from heavy to very heavy (?0.69 g/cm³). The total volumetric shrinkage values are low, less than 10%, for all species. The parallel compression strength and the shear strength for all the species indicate a very resistant wood (?46.93 MPa and ?18.35 MPa, respectively). Brinell hardness was higher than 5 kg/mm² in all cases. The species with less content of extractives is P. ruscifolia (approximately 9% of anhydrous mass) whereas A. aroma was the one with the greatest content (approximately 25% of anhydrous mass in the heartwood).     

Restoration of zooplankton in a small acidified mountain lake (Plešné Lake,Bohemian Forest) by reintroduction of key species

Atmospheric acidification brought about the extinction of several species of planktonic Crustacea in lakes of the Bohemian Forest (?umava, Böhmerwald). The recent reversal in chemical parameters of lake water towards pre-acidification values raises the question whether the return of missing species is possible. We have designed a bioassay to test water from different lakes for the survival of Daphnia longispina, which inhabited the lakes under study but disappeared from those most affected by acidification. The bioassay showed the best survival in water from Ple?né Lake, one of those where Daphnia and Cyclops became extinct. A large-scale experiment was conducted by transplanting Daphnia longispina (Crustacea, Cladocera) and Cyclops abyssorum (Crustacea, Copepoda) from Prá?ilské Lake, where they have survived until the present, to Ple?né Lake. One year later, a successful reintroduction was confirmed for Cyclops abyssorum.     

Trophic level delineation and resource partitioning in a South African afromontane forest bird community using carbon and nitrogen stable isotopes

Southern African forests are naturally fragmented yet hold a disproportionately high number of bird species. Carbon and nitrogen stable isotopes were measured in feathers from birds captured at Woodbush (n = 27 species), a large afromontane forest in the eastern escarpment of Limpopo province, South Africa. The δ¹³C signatures of a range of forest plants were measured to categorise the food base. Most plants sampled, including two of five grass species, had δ¹³C signatures typical of a C₃ photosynthetic pathway (?29.5 ± 1.9‰). Three grass species had a C₄ signature (?12.0 ± 0.6‰). Most bird species had δ¹³C values representing a predominantly C₃‐based diet (?24.8‰ to ?20.7‰). δ¹⁵N values were as expected, with higher levels of enrichment associated with a greater proportion of dietary animal matter. The cohesive isotopic niche defining most species (n = 22), where the ranges for δ¹³C and δ¹⁵N were 2.4‰ and 3.4‰, respectively, highlight the difficulties in understanding diets of birds in a predominantly C₃‐based ecosystem using carbon and nitrogen stable isotopes. However, variation in isotopic values between and within species provides insight into possible niche width and the use of resources by different birds within a forest environment.     

The enthalpy of oxidation of flavin mononucleotide: Temperature dependence of in vitro bacterial luciferase bioluminescence

The enthalpy of the bioluminescent reaction

$\text{FMNH}{}_2\text{+ RCHO + O}{}_2\text{→}\text{luciferase}\text{FMN + RCOO + H}{}_3\text{O}{}^{\mathrm{+}}\text{+ hv}$

has been studied by direct calorimetric methods. Bacterial luciferase, isolated from Beneckea harveyi (formerly strain MAV) has been used to catalyze the oxidation of reduced flavin mononucleotide (FMNH₂) and a long chain aliphatic aldehyde (dodecanal, RCHO) by molecular oxygen to give the indicated products and blue-green light. The enthalpy measured for this process was found to be ΔH_L = ?338.9 k.J (mol FMN)^?1 (?81.0 kcal) at 25.00 °C and ?402.9 kJ (mol FMN)^?1 (?96.3 kcal) at 7.00 °C. Calculations based on redox electrode potentials indicate a corresponding value of the free energy change, ΔG_L = ?464.8 kJ (mol FMN)^?1 (?111.1 kcal), at 25 °C. Measurements were performed in 0.15 m phosphate buffer, pH 7.0 and the values were arrived at by correcting the observed heats for the heat associated with the autoxidation process: FMNH₂ + O₂ ? FMN + H₂O₂; ΔH_D = ?158.5 kJ (mol FMN)^?1 (?37.8). These data and a detailed thermodynamic analysis have demonstrated the need for two parameters, referred to as the intrinsic free energy, ΔG₁, and intrinsic enthalpy, ΔH₁, which are functionally defined by the relations ΔG_I = ΔG_L ? uhvΔH_I = ΔH_L ? uhv, where u is the quantum yield of the reaction expressed in einsteins mole^?1.These parameters reflect the thermochemistry of the bioluminescent reaction corrected for emitted photons. Thus, they are useful for comparing the thermochemistry of a chemiluminescent process. Their values for the bacterial luciferase system at 25 °C and pH 7.0 are ?391.6 and ?266.9 kJ (mol FMN)^?1 (?93.6 and ?63.8 kcal), respectively, assuming a value of 0.3 for the quantum yield. The calorimetric data also suggest the existence of a long-lived species which persists after photon emission.     

Etagement des algues et des invertébrés sessiles dans l'Archipel de Glénan

The Glénan Archipelago was investigated, from the surface to a depth of 60 m, byScuba diving. Four distinct communities (“peuplements”) have been determined that fit into the system of zonation defined by the Genoa Symposium (1957). The results are compared with observations made in the English Channel (Cabioch 1968) and in the Atlantic Ocean (Glemarec 1971). Since illumination depends upon the angle of the rocky face, photophilic species exhibit a maximum range when the rock surfaces are horizontal. The sublittoral zone is dominated by laminarians. They form two distinct communities with the animal and vegetal species they overshadow. The upper community is characterized byLaminaria digitata (Linné)Lamouroux (horizontal rocky face from 0 to 6 m; vertical rocky face from +0.5 to ?3 m). The lower cummunity, characterized byL. hyperborea (Gunner)Foslie has a greater vertical extension (horizontal: ?6 m to ?26 m; vertical: ?3 to ?30 m). The circalittoral zone is colonized by several animal species growing in a ramified manner: the upper community is characterized by Axinellidae (?30 m), the lower byDendrophyllia cornigera (Lamarck) (?55 to ?60 m). Upright algae disappear somewhere between ?40 and ?47 m.     

Electrophoresis of DNA in agarose gels. II. Effects of loading mass and electroendosmosis on electrophoretic mobilities

We have investigated several experimental factors which affect the accurate determination of electrophoretic mobilities of circular and linear DNAs in agarose gels. We demonstrate that: (1) The mobility of individual DNA species is affected by the total mass in the sample loaded. The increased mobility and band distortion observed become apparent when the DNA mass exceeds approximately 0.2 μg per 0.15 cm² of surface area in the loading well. (2) The migration velocity of a given DNA species depends on the coefficient of electroendosmosis (?m_r) of the agarose preparations used. In the range 0.081 ≤ ?m_r ≤ 0.441, the DNA migration velocity is proportional to (?m_r)^?0.5.     

Nitrogen fertilisation reduces grass-induced N2 fixation of tree seedlings from semi-arid savannas

Aims

Coexistence of trees and grasses in nutrient-poor arid savannas may result in competition for soil N. While grasses may be more effective than woody plants in acquiring N from the soil, some leguminous woody species rely on N₂ fixation. We assessed the role of N₂ fixation in the N-budget of Acacia mellifera seedlings by varying N supply and grass competition.

Methods

The contribution of N₂ fixation to the N-budget of Acacia mellifera seedlings with varying N supply and grass competition was determined by measuring growth, nutrient concentrations, and ¹⁵N values.

Results

Tree seedlings were 4-fold taller and had 20-fold more biomass in the absence of grass. Tree foliar δ¹⁵N was lower with (?0.25?±?0.2‰, n?=?9) than without grasses (5.2?±?0.1‰, n?=?64). The contribution of N₂-fixation to the N budget decreased with increasing N supply. Greater reliance on N₂-fixation by trees in the presence of grasses did not result in greater biomass accumulation or tissue [N] relative to tree seedlings grown without grass competition. Tree seedlings competing with grass had significantly more negative δ¹³C (?29.5?±?0.6‰) than seedlings without grass competition (?28.8‰?±?0.5‰).

Conclusions

Induction of N₂-fixation by grass may have resulted from competition for nutrients. N₂-fixation enables tree seedlings to compensate for limited soil N and survive grass competition at a critical and vulnerable developmental stage of germination and establishment.     

Isolation and Characterization of Fruit-specific Promoters ACS4 and EXP1 from Tomato (Solanum lycopersicum L)

Promoter engineering in plants holds a great promise for understanding complexity of genetic regulatory system in response to specific internal and external cues and for crop improvement. In the present investigation, we report characterization of two fruit-specific promoters SIACS4 and SIEXP1 that were isolated from tomato (Solanum lycopersicum L cv Pusa Ruby). In silico analysis of the cloned promoter sequences revealed the presence of a seed-specific cis-element in SIACS4 and several putative seed, embryo and endosperm-specific cis-elements in SIEXP1 in addition to fruit-specific ethylene responsive regulatory elements. The fruit- and seed-specific expression of both the promoters was analyzed in transgenic tomato lines expressing the promoter:: GUS fusion constructs. The SIACS4 promoter (?1 to ?373) showed GUS activity restricted specifically to flower buds and seeds in fruits. On the contrary, the SIEXP1 promoter (?1 to ?769) showed high level of expression in seeds as compared to fruit tissues at different stages of fruit ripening. No GUS expression was observed in leaves satisfying the fruit-specific nature of both the promoters. Based on deletion analysis, minimal promoters SIACS4DL2 (?1 to ?126) and SIEXP1DL1 (?1 to ?254) were identified which can be used to drive tissue-specific expression of transgenes for introducing traits of agronomic importance such as resistance to fruit borer and for enhancing both nutritional and keeping quality of tomato fruits.     

Growth and photosynthetic responses of soybean seedlings to maize shading in relay intercropping system in Southwest China

Intercropping, a traditional and worldwide cropping practice, has been considered as a paradigm of sustainable agriculture based on complementary mechanisms among different crop species. Soybean (Glycine max) is widely relay intercropped about 60 days before maize (Zea mays) harvest in Southwest China. However, shade caused by tall maize plants may be a limiting factor for soybean growth at a seedling stage. In field research, we studied the ecophysiological responses of two widely cultivated soybean varieties [Gongqiudou494-1 (GQD) and Gongxuan 1 (GX)] to maize shading in a relay intercropping system (RI) compared with monocropped soybean plants (M). Our results showed that soybean seedlings intercropped with maize exhibited significantly downregulated net photosynthetic rate (P _N) (?38.3%), transpiration rate (?42.7%), and stomatal conductance (?55.4%) due to low available light. The insignificant changes in intercellular CO₂ concentration and the maximum efficiency of PSII photochemistry suggested that the maize shading-induced depressions in P _N were probably caused by the deficiency of energy for carbon assimilation. The significantly increased total chlorophyll (Chl) content (+27.4%) and Chl b content (+52.2%), with lowered Chl a/b ratios (?20.5%) indicated soybean plants adjusted their light-harvesting efficiency under maize shading condition. Biomass and leaf area index (LAI) of seedlings under RI decreased significantly (?78.7 and ?71%, respectively) in comparison with M. Correlation analysis indicated the relative reduction in biomass accumulation was caused by the decline in LAI rather than P _N, it affected negatively the final yields of soybean (32.8%). Cultivar-specific responses to maize shading were observed in respects of LAI, biomass, and grain yield. It indicated that GX might be a better cultivar for relay intercropping with maize in Southwest China.     

Effects of freeze-thaw stress on bacterial populations in soil microcosms

To test the effect of freezing on soil biota, isolated from the shortgrass prairie of northeastern Colorado, a series of experiments were performed using gnotobiotic soil microcosms.Pseudomonas paucimobilis was used to examine the effects of freezing on bacteria of different growth stages. Secondly, the effect of multiple freeze-thaw cycles was tested on an assemblage of bacterial species. Lastly, the effect of freezing on predator-prey interactions was studied usingP. paucimobilis and an amoebal predator,Acanthamoeba polyphaga. A temperature of ?9°C was not detrimental toP. paucimobilis at any growth stage. A single severe freeze-thaw cycle (?27°C to 23°C) resulted in 40–60% mortality ofP. paucimobilis and the mixed bacteria, although additional freezing events did not reduce the populations further. Multiple freeze-thaw cycles (?9°C to 23°C) gave 40–60% mortality ofP. paucimobilis and the mixed bacteria. Predator-prey population cycles were possibly desynchronized by freeze-thaw events.     

The composition of the chitinolytic microbial complex and its effect on chitin decomposition at various humidity levels

The dynamics of assimilation of chitin by soil microorganisms (primarily prokaryotes) as a source of carbon and nitrogen has been determined by gas chromatography and fluorescence microscopy. The highest rates of chitin decomposition in chernozem were detected at humidity levels corresponding to the pressure of soil moisture (P) of ?1.4 atm. The rate of microbial consumption of chitin is three times higher than that of the carbon of soil organic matter. Fluorescence microscopy revealed that an increase in the pressure of soil moisture from P = ?10 atm to P = ?0.7 atm resulted in a considerable increase in the proportion of the specific surface of mycelial bacteria (actinomycetes).     

Photosynthetic and wood anatomical responses of Tamarix africana Poiret to water level reduction after short-term fresh- and saline-water flooding

In the Mediterranean basin, Tamarix spp. constitute important populations along rivers and sea coasts, and might be primarily subjected to water level fluctuations and salinization, as a consequence of global climate change. Here, we analyze leaf gas exchange and xylem anatomy during a water level decrease below the soil surface after short-term flooding with fresh- and saline-water (200?mM) in order to predict Tamarix africana Poiret responses under future environmental conditions. Fresh-water level reduction negatively affected stomatal conductance (?56.3?%), but only when water decreased to the lowest level (15?cm below the soil surface). No effects on assimilation rates and xylem vessel dimensions occurred. Under saline conditions, the rate of the water level decrease was lower compared to the non-saline treatment, as stomatal conductance was negatively affected by salinity (?59.5?%) and significantly declined over time. Moreover, decreases in mean xylem vessel area (?51.3?%), assimilation rates (?52.2?%) and stomatal conductance (?76.0?%) were also observed compared to the control, indicating both an osmotic stress and a toxic effect of NaCl on leaf gas exchange. These leaf responses were probably induced by greater belowground-root salt absorption and transport compared to previous flooding conditions, as confirmed by the increase in salt excretion (+473.2?%). The results emphasize the survival risk of Tamarix spp. to water level variation under both saline and non-saline conditions, and the need of management practices focused on the conservation of these populations.     

Improvement of expression level of keratinase Sfp2 from Streptomyces fradiae by site-directed mutagenesis of its N-terminal pro-sequence

The keratinase Sfp2, produced by Streptomyces fradiae var. k11, is a serine alkaline protease first synthesized as pre-pro-mature precursor, of which the N-terminal propeptide must be autocatalytically cleaved on the C-terminal of P1 amino acid to produce mature enzyme. Single amino acid substitutions were introduced at positions ?1 and ?2 to improve the expression level of mature Sfp2. The specific activity of L(?1)F mutant (48935 U/mg) was nine times that of wild-type Sfp2, whereas the mutants L(?1)D, L(?1)G, L(?1)H, K(?2)E, and K(?2)L had 2–52 % of the specific activity of wild-type. The yield of mature Sfp2 of L(?1)F mutant was estimated to be 800 μg/mg total protein and 112 mg/l culture supernatant, nine and twice that of wild-type, respectively. The L(?1)F mutant exhibited similar enzymatic properties to wild-type.     

Stable isotopic (δ13C and δ18O) signatures of biogenic calcretes marking discontinuity surfaces: a case study from Upper Cretaceous carbonates of central Dalmatia and eastern Istria,Croatia

Biogenic calcretes associated with a regional Cretaceous to Paleogene subaerial unconformity and an intraformational composite (polygenic) surface in Upper Cretaceous intra-platform peritidal successions in central Dalmatia and eastern Istria, Croatia (Adriatic-Dinaridic Carbonate Platform), were analyzed for their δ¹³C and δ¹⁸O signatures in order to provide insight into the conditions of subaerial exposure and calcrete development. The distinctly negative δ¹³C signatures of biogenic calcretes marking the regional subaerial unconformity differ considerably from the δ¹³C values of the host marine limestones. This indicates carbon isotope exchange of primary marine CaCO₃ with CO₂ released by root and rhizomicrobial respiration and subsequent precipitation of pedogenic calcrete. The range of δ¹³C (from ?13.1 to ?8.2 ‰ Vienna PeeDee Belemnite standard, VPDB) and δ¹⁸O (from ?10.1 to ?6.1 ‰ VPDB) values of calcretes are similar to those reported from calcretes elsewhere, and the δ¹³C values of biogenic calcretes with typical Microcodium aggregates (?13.1 to ?12.3 ‰ VPDB) at the ?ibenik locality are very close to, or at the lower limit of, values for soil carbonates formed in isotopic equilibrium with soil CO₂. These values are expected for authigenic pedogenic carbonates formed under the influence of C₃ plant communities, without influence from heavier carbon from pre-existing carbonate and lack of input of atmospheric CO₂. Such low δ¹³C values support the interpretation of Microcodium aggregates as being precipitated under a direct biological control within the soil, although the relationship between formation mechanisms and stable isotope signatures of Microcodium needs further investigation. The δ¹³C values (?4.4 to ?3.6 ‰ VPDB) of rhizogenic calcretes formed inside firmground Thalassinoides burrows of the composite surface at the ?ibenik locality are more negative than the δ¹³C values of the host marine limestones, which confirms that the composite surface went through a phase of meteoric pedo(dia)genesis. However, the overall δ¹³C values of calcretes are less negative than expected, which might reflect contamination from associated primary marine carbonate. This study represents the first detailed stable isotope investigation of calcretes from carbonate successions of the External Dinarides, and the results may be applied to discontinuities present in other shallow-water carbonate rock successions.     

Wild blueberry (Vaccinium angustifolium) consumption improves inflammatory status in the obese Zucker rat model of the metabolic syndrome

The metabolic syndrome (MetS) is a major public health problem in the United States. Chronic inflammation is a critical component of the MetS, leading to dramatically increased risk of type II diabetes and cardiovascular disease.This study investigates the ability of a wild-blueberry-enriched diet to improve the proinflammatory status associated with MetS in the obese Zucker rat (OZR). Circulating levels of key inflammatory markers and their expression in the liver and abdominal adipose tissue were examined in OZR and its genetic control, the lean Zucker rat (LZR), after feeding a control or an 8% wild blueberry diet (WB) for 8 weeks from age 8 to 16 weeks.In the OZR, WB consumption resulted in decreased plasma concentrations of tumor necrosis factor (TNF)-α (?25.6%, P<.05), interleukin (IL)-6 (?14.9%, P<.05) and C-reactive protein (CRP) (?13.1%, P<.05) and increased adiponectin concentration (+21.8%, P<.05). Furthermore, expression of IL-6, TNF-α and nuclear factor (NF)-kB was down-regulated in both the liver (?65%, ?59% and ?25%, respectively) and the abdominal adipose tissue (?64%, ?52% and ?65%), while CRP expression was down-regulated only in the liver (?25%). In the abdominal adipose tissue, similar trends were also observed in LZR following WB treatment, with decreased liver expression of NF-kB, CRP, IL-6 and TNF-α (?24%, ?16%, ?21% and ?50%) and increased adiponectin expression (+25%).Results of this study suggest that wild blueberry consumption exerts an overall anti-inflammatory effect in the OZR, a model of the metabolic syndrome.     

Effect of water stress on the enzymes of nitrate metabolism in two Brassica species

Brassica juncea (drought susceptible) and B. campestris (drought tolerant) were germinated under simulated water stress created by polyethylene glycol (MW 6000). The two species showed characteristic differences in dry weight, nitrate reductase, aspartate amino transferase, alanine aminotransferase, glutamate dehydrogenase and free proline accumulation in the embryo axis under water stress. Stress resulted in the decreased activities of these enzymes and the decrease was more in B. juncea than in B. campestris. In both species, protein content was higher under stress. In B. juncea, a 12-fold increase in free proline occurred as compared to a 7-fold increase in B. campestris at ?6 atm osmotic potential.     

A global perspective of ground level, ‘ambient’ carbon dioxide for assessing the response of plants to atmospheric CO2

For most studies involving the response of plants to future concentrations of atmospheric carbon dioxide (CO₂), a current concentration of 360–370 μatm is assumed, based on recent data obtained from the Mauna Loa observatory. In the present study, average seasonal diurnal values of ambient CO₂ obtained at ground level from three global locations (Australia, Japan and the USA) indicated that the average CO₂ (at canopy height) can vary from over 500 μatm at night to 350 μatm during the day with average 24‐h values ranging from 390 to 465 μatm. At all sites sampled, ambient CO₂ rose to a maximum value during the pre‐dawn period (03.00–06.00 hours); at sunrise, CO₂ remained elevated for several hours before declining to a steady‐state concentration between 350 and 400 μatm by mid‐morning (08.00–10.00 hours). Responses of plant growth to simulations of the observed variation of in situ CO₂ were compared to growth at a constant CO₂ concentration in controlled environment chambers. Three diurnal patterns were used (constant 370 μatm CO₂, constant 370 during the day (07.00–19.00 hours), high CO₂ (500 μatm) at night; or, high CO₂ (500 μatm) at night and during the early morning (07.00–09.00 hours) decreasing to 370 μatm by 10.00 hours). Three plant species ? soybean (Glycine max, L (Merr.), velvetleaf (Abutilon theophrasti L.) and tomato (Lycopersicon esculentum L.) ? were grown in each of these environments. For soybean, high night‐time CO₂ resulted in a significant increase in net assimilation rate (NAR), plant growth, leaf area and biomass relative to a constant ambient value of CO₂ by 29 days after sowing. Significant increases in NAR for all three species, and significant increases in leaf area, growth and total biomass for two of the three C3 species tested (velvetleaf and soybean) were also observed after 29 days post sowing for the high night/early morning diurnal pattern of CO₂. Data from these experiments suggest that the ambient CO₂ concentration experienced by some plants is higher than the Mauna Loa average, and that growth of some agricultural species at in situ CO₂ levels can differ significantly from the constant CO₂ value used as a control in many CO₂ experiments. This suggests that a reassessment of control conditions used to quantify the response of plants to future, elevated CO₂ may be required.