Multicomponent Investigations of the Hydrogen Peroxide- and Hydroxyl Radical-Scavenging Antioxidant Capacities of Biofluids: The Roles of Endogenous Pyruvate and Lactate Relevance to Inflammatory Joint Diseases

High field proton (¹H) nuclear magnetic resonance (NMR) analysis of biofluids (healthy human blood sera and inflammatory knee-joint synovial fluids) has been employed to evaluate the hydrogen peroxide (H₂O₂)- and hydroxyl radical (°OH)- scavenging antioxidant capacities of a range of polar, low-molecular-mass endogenous metabolites therein. Data obtained indicate that consumption of H₂O₂ by pyruvate (generating acetate and CO₂ via an oxidative decarboxylation reaction) and °OH radical by lactate (generating pyruvate, and subs quently acetate and CO₂) may serve to protect alternative biofluid components (e.g., macromolecules) against reactive oxygen species-mediated oxidative damage in vivo. The mechanistic, physiological and potential therapeutic implications of these results are discussed with special reference to inflammatory joint diseases.     

Preventing metal-mediated oxidative DNA damage with selenium compounds

Copper and iron are two widely studied transition metals associated with hydroxyl radical (˙OH) generation, oxidative damage, and disease development. Because antioxidants ameliorate metal-mediated DNA damage, DNA gel electrophoresis assays were used to quantify the ability of ten selenium-containing compounds to inhibit metal-mediated DNA damage by hydroxyl radical. In the Cu(I)/H(2)O(2) system, selenocystine, selenomethionine, and methyl-selenocysteine inhibit DNA damage with IC(50) values ranging from 3.34 to 25.1 μM. Four selenium compounds also prevent DNA damage from Fe(II) and H(2)O(2). Additional gel electrophoresis experiments indicate that Cu(I) or Fe(II) coordination is responsible for the selenium antioxidant activity. Mass spectrometry studies show that a 1?:?1 stoichiometry is the most common for iron and copper complexes of the tested compounds, even if no antioxidant activity is observed, suggesting that metal coordination is necessary but not sufficient for selenium antioxidant activity. A majority of the selenium compounds are electroactive, regardless of antioxidant activity, and the glutathione peroxidase activities of the selenium compounds show no correlation to DNA damage inhibition. Thus, metal binding is a primary mechanism of selenium antioxidant activity, and both the chemical functionality of the selenium compound and the metal ion generating damaging hydroxyl radical significantly affect selenium antioxidant behavior.     

Effect of carbon dioxide on antioxidant enzymes and ginsenoside production in root suspension cultures of Panax ginseng

The aim of this study was to investigate the effect of CO₂ at various concentrations (1, 2.5 and 5%) on antioxidant enzymes and ginsenoside accumulation in Panax ginseng roots in 5 l airlift bioreactors (working volume 4 l). One and 2.5% CO₂ was beneficial for root biomass accumulation, but 5% CO₂ decreased the biomass. Ginsenoside concentration decreased with increasing concentration of CO₂. No significant difference was observed in the malondialdehyde (MDA) content and lipoxygenase (LOX) activity between respective controls and CO₂ treated roots. Antioxidant enzymes such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione reductase (GR), catalase (CAT), guaiacol peroxidase (G-POD) including reduced ascorbate and total glutathione were induced in CO₂ exposed roots which emphasized the protective role of antioxidants against CO₂ induced stress. Superoxide dismutase activity (SOD) which was induced after 15 days was significantly inhibited after 45 days. Glutathione-S-transferase (GST) and glutathione peroxidase (GPX) activities also increased when the roots were subjected to 1 and 2.5% CO₂ compared to the respective controls but not at 5%. A higher reduced ascorbate to oxidized (ASC/DHA) ratio in CO₂ treated root indicates the plant's ability to tolerate CO₂ stress. These observations suggest that an increase in antioxidant enzymes may affect a defense response to the cellular damage induced by CO₂. Probably, this increase could not stop the deleterious effects of CO₂ concentration on ginsenoside concentration, but reduced stress severity and thereby allowing root growth to occur.     

CO2加富对UV-B辐射胁迫下亚心形扁藻光合作用和膜脂过氧化以及抗氧化酶活性的影响

在CO2浓度分别为当今CO2浓度(360 μL/L)和加富浓度(5 000 μL/L)条件下,研究了UV-B胁迫对亚心形扁藻(Platymonas subcordiformis(Wille)Hazen)的光合作用、膜脂过氧化和抗氧化酶活性的影响.实验结果表明:(1)UV-B单独作用下,亚心形扁藻的干重、光合速率、叶绿素a(Chl a)和类胡萝卜素(Car.)含量显著降低,CO2加富单独作用下,亚心形扁藻的干重和光合速率显著升高,叶绿素a和类胡萝卜素含量与对照相比没有显著变化,而UV-B与CO2共同作用则使亚心形扁藻的干重和光合速率与对照相比没有显著变化,叶绿素a和类胡萝卜素含量显著降低.(2)UV-B单独作用和CO2加富单独作用都使可溶性蛋白含量显著降低,UV-B与CO2共同作用下的可溶性蛋白含量比UV-B单独作用的要高.高CO2对藻的可溶性蛋白含量的变化在很大程度上归因于Rubisco蛋白的降低.(3)UV-B单独作用下,O-.2产生速率、H2O2含量和MDA含量显著升高,而CO2加富单独作用下,O-.2产生速率、H2O2含量和MDA含量显著降低,与UV-B单独作用相比,UV-B与CO2共同作用使O-.2产生速率、H2O2含量和MDA含量显著降低.说明CO2加富可以减少活性氧对亚心形扁藻的氧化胁迫,同时减少UV-B对亚心形扁藻的膜脂过氧化伤害.(4)UV-B单独作用下,SOD、POD、CAT、GR和GPx活性显著升高,高CO2单独作用使SOD、POD和GR活性显著降低,而CAT和GPx活性与对照相比稍有所降低,但降低不明显,而UV-B与CO2共同作用则使SOD、POD、CAT、GR和GPx活性比UV-B单独作用少得多.结果表明,高CO2对UV-B胁迫所造成的氧化胁迫具有一定的改善作用,因此CO2浓度升高可能对增强海洋微藻的抗逆能力有利.     

Potential of Bioaugmentation and Biostimulation for Enhancing Intrinsic Biodegradation in Oil Hydrocarbon-Contaminated Soil

Studies were conducted using a 10-chamber Micro-Oxymax (Columbus, OH, USA) respirometer to determine the effect of bioaugmentation and biostimulation (by diverse ways of O₂ supply) on enhancing biodegradation of oil hydrocarbons to reduce risk at a former military airport in Kluczewo, Poland. Indigenous or exogenous bacteria bioaugmentation was used to degrade hydrocarbons. Aerated water and/or aqueous solutions of H₂O₂ or KMnO₄ were used to supply O₂. The intrinsic and enhanced biodegradation was evaluated by the O₂ uptake and CO₂ production rates obtained using a linear regression of the cumulative O₂ uptake and CO₂ production curves. Generally, in all cases biodegradation rates enhanced by bioaugmentation were two to four times higher than the rates of intrinsic biodegradation. Moreover, application of indigenous bacteria was more efficient in comparison to the exogenous consortia. The highest CO₂ production rates were achieved when aqueous solution of KMnO₄ was applied, as the increase of CO₂ production rates were about 71% to 97% higher compared to a control. The aqueous solution of H₂O₂ did not cause any significant improvement of the biodegradation rates. Compared to a control, the addition of aerated water resulted in a decrease of CO₂ production rates. Most probably the excessive soil moisture could reduce the air-filled porosity and, consequently, the oxygen contents in soil.     

In vivo relevance for photoprotection by the vitamin D rapid response pathway

Vitamin D is produced by exposure of 7-dehydrocholesterol in the skin to UV irradiation (UVR) and further converted in the skin to the biologically active metabolite, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) and other compounds. UVR also results in DNA damage producing cyclobutane pyrimidine dimers (CPD). We previously reported that 1,25(OH)₂D₃ at picomolar concentrations, protects human skin cells from UVR-induced apoptosis, and decreases CPD in surviving cells. 1,25(OH)₂D₃ has been shown to generate biological responses via two pathways—the classical steroid receptor/genomic pathway or a rapid, non-genomic pathway mediated by a putative membrane receptor. Whether the rapid response pathway is physiologically relevant is unclear. A cis-locked, rapid-acting agonist 1,25(OH)₂lumisterol₃ (JN), entirely mimicked the actions of 1,25(OH)₂D₃ to reduce fibroblast and keratinocyte loss and CPD damage after UVR. The effects of 1,25(OH)₂D₃ were abolished by a rapid-acting antagonist, but not by a genomic antagonist. Skh:hr1 mice exposed to three times the minimal erythemal dose of solar-simulated UVR and treated topically with 1,25(OH)₂D₃ or JN immediately after UVR showed reduction in UVR-induced UVR-induced sunburn cells (p < 0.01 and <0.05, respectively), CPD (p < 0.01 for both) and immunosuppression (p < 0.001 for both) compared with vehicle-treated mice. These results show for the first time an in vivo biological response mediated by a rapid-acting analog of the vitamin D system. The data support the hypothesis that 1,25(OH)₂D₃ exerts its photoprotective effects via the rapid pathway and raise the possibility that other D compounds produced in skin may contribute to the photoprotective effects.     

An Improved Squash-Slide Freezer

Liquid CO₂ is suited to more widespread use than the solid form, through its more extensive availability and much longer “shelf-life” in storage. Earlier models of devices for freezing squash-bearing slides used a chilled metal plate, but Bowen's design (Stain Techn., 31: 87-90, 1956) allowed the CO₂ to expand below and in direct contact with the slide. In the present model, the jet of CO₂ is directed downward within an insulated chamber which holds the slide. Advantages are: economy in the use of CO₂, facilitation in handling of slides, and quiet operation. CO₂ cylinders with built-in riser pipes are recommended for use in the vertical position.     

Effect of hydroxylated fullerene C60(OH)25 on macrophage plasma membrane integrity

Our study has shown that the damaging effect of hydroxylated fullerene C60(OH)25 on mouse peritoneal macrophage plasma membranes increased when we enlarged the concentration of fullerene in the incubation media (from 0.005 to 0.5 mg/ml), the incubation temperature (from 22 degrees C to 37 degrees C) and the time of incubation (from 30 to 90 min). In conditions of the H2O2-induced membrane damage, fullerene was observed to intensify the H2O2-induced damaging effect at a concentration of 0.05 mg/ml and reduce it at a concentration of 0.5 mg/ml. In conditions of the UV-induced membrane damage, it was discovered that the damaging effect of UV increased when C60(OH)25 nanoparticles were added to the incubation media before irradiation and decreased when they were added after irradiation. Eventual participation of ROS in damaging effects of C60(OH)25 was discussed.     

Reduction of oxygen by the electron transport chain of chloroplasts during assimilation of carbon dioxide

In photosynthetically competent chloroplasts from spinach the quantum requirements for oxygen evolution during CO₂ reduction were higher, by a factor often close to 1.5, than for oxygen evolution during reduction of phosphoglycerate. Mass spectrometer experiments performed under rate-limiting light indicated that an oxygen-reducing photoreaction was responsible for the consumption of extra quanta during carbon dioxide assimilation. Uptake of ¹⁸O₂ during reduction of CO₂ was considerably higher than could be accounted for by oxygen consumption during glycolate formation and by the Mehler reaction of broken chloroplasts which were present in the preparations of intact chloroplasts. The oxygen reducing reaction occurring during CO₂ assimilation resulted in the formation of H₂O₂. This was indicated by a large stimulation of CO₂ reduction by catalase, but not of phosphoglycerate reduction. Catalase could be replaced as a stimulant of photosynthesis by dithiothreitol or ascorbate, compounds known to react with superoxide radicals. There was no effect of dithiothreitol and ascorbate on phosphoglycerate reduction. A main effect of superoxide radicals and/or H₂O₂ was shown to be at the level of phosphoglycerate formation. Evidence for electron transport to oxygen was also obtained from ¹⁴CO₂ experiments. The oxidation of dihydroxyacetonephosphate during a dark period or after addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the light was studied. The results indicated a link between the chloroplast pyridine nucleotide system and oxygen. Oxygen reduction during photosynthesis under conditions where light is rate limiting is seen as important in supplying the ATP which is needed for CO₂ reduction but is not provided during electron transport to NADP. A mechanism is discussed which would permit proper distribution of electrons between CO₂ and oxygen during photosynthesis.     

Differences between cysteine and homocysteine in the induction of deoxyribose degradation and DNA damage

The effect of two naturally occurring thiols, such as cysteine and homocysteine, has been examined for their ability to induce deoxyribose degradation and DNA damage. Copper(II) ions have been added to incubation mixtures and oxygen consumption measurements have been performed in order to correlate the observed damaging effects with the rate of metal catalyzed thiol oxidation. Ascorbic acid plus copper has been used as a positive control of deoxyribose and DNA oxidation due to reactive oxygen species. Cysteine or homocysteine in the presence of copper ions induce the degradation of deoxyribose and the yield of 8-hydroxy-2'-deoxyguanosine (8-OHdG), although important differences are observed between the two thiols tested, homocysteine being less reactive than cysteine. DNA cleavage is induced by cysteine in the presence of copper(II) ions but not by homocysteine. Catalase and thiourea, but not superoxide dismutase (SOD), were shown to inhibit the damaging effects of cysteine on deoxyribose or DNA suggesting that H(2)O(2) and *OH radicals are responsible for the observed induced damage. The results indicate that there are differences between the damaging effects of the two thiols tested towards deoxyribose and DNA damage. The pathophysiological importance will be discussed.     

Microalgae-mediated chemicals production and wastes removal

Biotechnology of microalgae has gained importance in recent years due to the development of new production and environmental technologies. Because their growth requires unexpensive substrates such as solar light and CO₂, microalgae can be used as cheap and effective biocatalysts to obtain high added-value compounds, from simple metabolites to complex molecules, i.e., chemicals, vitamins, carotenoids, pigments, or polysaccharides. During productive processes, the algal biomass formed may be used as a food source like proteins. On the other hand, microalgae can also be employed in contaminant bioelimination processes especially for nitrogen, phosphorus, or sulfur compounds. Particularly relevant is their use for heavy metal removal from wastewaters; upon enriching the biomass in the metal, they can be recovered, thereby providing economic advantages.

The use of immobilized microalgae in these processes is very adequate and offers significant advantages in bioreactors.     

大气CO2浓度升高对植物、植食性昆虫及其天敌的影响研究进展

自世界工业革命以来,化石燃料的大量使用以及人类对自然环境的过度破坏,致使大气CO₂浓度不断升高.研究大气CO₂浓度升高介导的农业生态系统内植物、植食性昆虫及其天敌的适应机制,对于阐明气候变化下农业害虫爆发危害规律,指导防控与减排具有重要意义.本文综述了大气CO₂浓度升高对农业生态系统中植物、植食性昆虫及天敌的影响,主要包括：1)相关研究方法的改进;2)大气CO₂浓度升高介导的寄主植物营养和次生代谢物质的变化;3)大气CO₂浓度升高对以植物为食的昆虫的个体生长发育、种群数量、行为的影响;4)天敌昆虫的生物学及捕食量与寄生率变化.最后对今后的研究方向进行了展望.     

昆虫对CO2感受机制的研究进展

CO2是空气的重要组分,它既是植物光合作用的主要原料之一,也可以作为环境中的化学信号直接影响昆虫的生命活动.CO2在双翅目、鞘翅目和鳞翅目昆虫的觅食行为中都发挥着重要作用;CO2可以影响蝇类和蛾类的产卵行为;CO2是社会性昆虫调节巢穴内气候条件的重要指标.对昆虫感受CO2的机制研究一方面可以拓展对昆虫化学感受体系的认知...     

大气CO2浓度升高对香蕉光合作用及光合碳循环过程中叶氮分配的影响

生长在高CO₂浓度(700±5μl·L^-1)1周的香蕉叶片,其光合速率(Pn,μmol·m^-2·s^-1)为5.14±0.32,较生长在大气CO₂浓度(356±301μl·L^-1)的高22.1%,而生长在较高CO₂浓度下8周,叶片Pn较生长在大气CO₂浓度的低18.1%,表现香蕉叶片对较长期高CO₂浓度的驯化和光合作用抑制.生长在高CO₂浓度的香蕉叶片有较低光下呼吸速率(R_d),而不包括光下呼吸的CO₂补偿点则变幅较小.最大羧化速率(Vcmax)和电子传递速率(J)分别较生长在大气CO₂浓度的低30.5%和14.8%,根据气体交换速率计算的表观量子产率(α,mol CO₂·mol^-1光量子),生长在较高CO₂浓度下8周的叶片为0.014±0.01,而生长在大气CO₂浓度下的为0.025±0.005.较高CO₂浓度下叶片的表观量子产率降低44%.光能转换效率electrons·quanta^-1)亦从0.203降低至0.136.生长在较高CO₂浓度下香蕉叶片的叶氮在Rubicos分配系数(PR)、叶氮在生物力能学组分分配系数(P_B)和叶氮在光捕组分的分配系数(P_L)均较生长在大气CO₂浓度低,表明在高CO₂浓度下较长期生长(8周)的香蕉叶片多个光合过程受抑制,光合活性明显降低.     

高CO2浓度对长白山阔叶红松林主要树种的影响

组成长白山阔叶红松林的主要树种红松、云杉、落叶松、大青杨、白桦、椴树、水曲柳和色木的幼树,盆栽于模拟自然光照和人工调节CO₂浓度为700μl·L^-1、400μl·L^-1的气室内两个生长季,以生长在400μl·L^-1下的幼树为对照组,在各自生长条件下测定,高CO₂浓度下生长的红松、云杉、落叶松、大青杨、白桦、椴树、水曲柳和色木的高生长比对照组的幼树提高10%～40%.高CO₂浓度的幼树与对照CO₂下的幼树相比各树种蒸腾速率升降不一,但水分利用效率均有不同程度的提高,不同树种的可溶性糖和叶绿素含量对高CO₂浓度反应不一,反映出幼树对高CO₂浓度适应的复杂性.长期高CO₂浓度环境下生长的阔叶树对CO₂变化反应较针叶树敏感,供试树种均发生光合驯化现象.     

亚热带天然阔叶林转换为杉木人工林对土壤呼吸的影响

采用静态箱-气相色谱法对浙江省临安市玲珑山风景区天然阔叶林和由天然阔叶林改造的杉木人工林的土壤呼吸进行1年的定位监测.结果表明：天然阔叶林和杉木人工林土壤CO₂排放速率均呈现一致的季节性变化规律即夏秋季高、冬春季低;天然阔叶林和杉木人工林土壤CO₂排放速率分别为20.0～111.3和4.1～118.6 mg C·m^-2·h^-1;天然阔叶林土壤CO₂年累积排放通量（16.46 t CO₂·hm^-2·a^-1）显著高于杉木人工林（11.99 t CO₂·hm^-2·a^-1）.天然阔叶林和杉木人工林土壤CO₂排放速率与土壤含水量均没有显著相关性,而与5 cm处土壤温度呈显著指数相关,Q₁₀值分别为1.44和2.97;天然阔叶林土壤CO₂排放速率与土壤水溶性碳（WSOC）含量无显著相关性,杉木人工林土壤CO₂排放速率与WSOC含量呈显著相关.天然阔叶林转换为杉木人工林显著降低了土壤CO₂排放,提高了土壤呼吸对环境因子的敏感性.
     

Synergistic Effects of Cu(II) and Dimethylammonium 2,4-Dichlorophenoxyacetate (U46 D Fluid) on PM2 DNA and Mechanism of Dna Damage

Dimethylammonium 2,4-dichlorophenoxyacetate (2,4-D · DMA) induced strand breaks in PM2 DNA when incubated with CuCl₂, whereas 2,4-D · DMA alone or CuCl₂ alone did not show any or only a negligible effect. The formation of single strand breaks increased linearly with time and concentration of 2,4-D · DMA. Neocuproine, a specific Cu(I) chelator totally prevented strand break formation. So did catalase (up to 100mM 2,4-D · DMA), but DMSO had only a small protective effect. 2,4-Dichlorophenol, CO₂ and formaldehyde were detected as reaction products of 2,4-D and CuCl₂. From these results a redox reaction of Cu(II) and 2,4-D is proposed, which could explain the DNA damaging properties of CuCl₂/2,4-D · DMA.     

大气CO2浓度升高对绿豆叶片光合作用及叶绿素荧光参数的影响

利用FACE系统在大田条件下通过盆栽试验研究了大气CO₂浓度升高\[CO₂浓度平均为（550±60) μmol·mol^-1\]对绿豆叶片光合生理和叶绿素荧光参数的影响.结果表明： 与对照\[CO₂浓度平均为（389±40）μmol·mol^-1左右\]相比,大气CO₂浓度升高使花荚期绿豆叶片净光合速率(P_n)和胞间CO₂浓度(C_i)分别升高11.7%和9.8%,气孔导度(G_s)和蒸腾速率(T_r)分别下降32.0%和24.6%, 水分利用效率(WUE)提高83.5%;在蕾期,CO₂浓度升高对绿豆叶片叶绿素初始荧光(F_o)、最大荧光（F_m）、可变荧光（F_v）、F_v/F_m和F_v/F_o没有显著影响;在鼓粒期,CO₂浓度升高使绿豆叶片F_o增加19.1%,F_m和F_v分别下降9.0%和14.3%,F_v/F_o和F_v/F_m分别下降25.8%和6.2%.表明大气CO₂浓度升高可能使绿豆生长后期光系统Ⅱ反应中心结构受到破坏,叶片的光合能力下降.     

Productivity of well-watered Panicum virgatum does not increase with CO2 enrichment

Aims Rising atmospheric CO₂ has been shown to increase aboveground net primary productivity (ANPP) in water-limited perennial grasslands, in part by reducing stomatal conductance and transpiration, thereby reducing depletion of soil moisture. However, the benefits of CO₂ enrichment for ANPP will vary with soil type and may be reduced if water limitation is low. Little is known about CO₂ effects on ANPP of Panicum virgatum, a perennial C₄ tallgrass and potential bioenergy crop. We hypothesized that if water limitation is minimized, (i) CO₂ enrichment would not increase P. virgatum ANPP because photosynthetic rates of this C₄ grass would not increase and because decreased transpiration at elevated CO₂ would provide little additional benefit in increased soil moisture and (ii) soil type will have little effect on P. virgatum CO₂ responses because of high overall soil moisture.Methods Growth and leaf physiology of P. virgatum cv. 'Alamo' were studied as plants established for 4 years on silty clay and clay soils along a 250 to 500 μl l -1 gradient in atmospheric CO₂ located in central Texas, USA. Plants were watered to replace evapotranspiration, fertilized with NO 3 NH 4 and P 2 O 5 and clipped to standard height during mid-season.Important findings ANPP increased through the third year of growth. Soil moisture (0–20 cm), ANPP, tiller numbers and leaf area index were 8–18% higher on the clay than on the silty clay soil. ANPP did not increase with CO₂ except in the planting year. However, biomass removed with clipping strongly increased with CO₂ in years 2 and 3, suggesting that CO₂ enrichment increased the early- to mid-season growth of establishing P. virgatum but not later regrowth or that of fully established plants. Furthermore, CO₂ enrichment differentially affected two components of ANPP in years 2 and 3, increasing tiller mass and reducing tiller numbers. This reallocation of resources in clipped P. virgatum suggested increased meristem limitation of productivity with CO₂ enrichment. CO₂ enrichment had little effect on photosynthesis but increasingly reduced stomatal conductance and transpiration as the plants established. As a result, water use efficiency became increasingly coupled to CO₂ as leaf area increased during establishment. These results suggest that for well-watered and clipped P. virgatum, ANPP differed between soil types, was not affected by CO₂ enrichment when fully established but interacted with clipping to alter allocation patterns during establishment. Soil type effects on ANPP-CO₂ responses will likely become more apparent when water is more limiting.     

CO2浓度升高条件下长白赤松幼苗种植密度对净光合速率的影响

The effect of Pinus sylvestriformis seedlings density on net photosynthetic rate was studied under elevated CO₂. Atmospheric CO₂ concentration was controlled in OTC (Open Top Chamber). The results showed that elevated CO₂ not only made net photosynthetic rates (NPRs) of two Pinus sylvestriformis seedlings densities increased,but also mitigated their intra-specific competition. Meanwhile,the difference of seedling NPRs between100 and 400 plant·m^-2 under 500 μmol·mol^-1 air CO₂ concentration was less than that under 350 μmol·mol^-1 with the same PARlevels. When air CO₂ concentration reached 700 μmol·mol^-1, the NPRs of seedlings under both planting densities were close to each other with the same PARlevels. The intra-specific competition was minimized under air CO₂ concentration of 700 μmol·mol^-1.