Effects of elevated CO₂ levels on root morphological traits and Cd uptakes of two Lolium species under Cd stress

Effects of elevated CO 2 levels on root morphological traits and Cd uptakes of two Lolium species under Cd stress 1 2 3 first-author 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:tangshir@hotmail.com †E-mail:tangshir@hotmail.com tangshir@hotmail.com 1 2 1 2 3 3 1 2 4 Centre for Research in Ecotoxicology and Environmental Remediation, Agro-environmental Protection Institute, Ministry of Agriculture, Tianjin 300191, China Centre for Research in Ecotoxicology and Environmental Remediation, Agro-environmental Protection Institute, Ministry of Agriculture, Tianjin 300191, China Open Key Laboratory of Agro-environment and Food Safety of Ministry of Agriculture, Tianjin 300191, China Open Key Laboratory of Agro-environment and Food Safety of Ministry of Agriculture, Tianjin 300191, China College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China Faculty of Health and Life Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK Faculty of Health and Life Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK This study was conducted to investigate the combined effects of elevated CO 2 levels and cadmium (Cd) on the root morphological traits and Cd accumulation in Lolium multiflorum Lam. and Lolium perenne L. exposed to two CO 2 levels (360 and 1000 μl/L) and three Cd levels (0, 4, and 16 mg/L) under hydroponic conditions. The results show that elevated levels of CO 2 increased shoot biomass more, compared to root biomass, but decreased Cd concentrations in all plant tissues. Cd exposure caused toxicity to both Lolium species, as shown by the restrictions of the root morphological parameters including root length, surface area, volume, and tip numbers. These parameters were significantly higher under elevated levels of CO 2 than under ambient CO 2 , especially for the number of fine roots. The increases in magnitudes of those parameters triggered by elevated levels of CO 2 under Cd stress were more than those under non-Cd stress, suggesting an ameliorated Cd stress under elevated levels of CO 2 . The total Cd uptake per pot, calculated on the basis of biomass, was significantly greater under elevated levels of CO 2 than under ambient CO 2 . Ameliorated Cd toxicity, decreased Cd concentration, and altered root morphological traits in both Lolium species under elevated levels of CO 2 may have implications in food safety and phytoremediation. Elevated CO 2 levels Lolium multiflorum Lam. Lolium perenne L. Root morphology Cd uptake Cd stress Introduction 1 The world’s industrialization has given rise to increases in the atmospheric carbon dioxide (CO 2 ) concentrations (from 280 to 380 μl/L) (IPCC, 2007 2007 ) and environmental pollution. Elevated levels of CO 2 and increased heavy metal concentrations in the agricultural environment potentially affect both plant growth and development, and pose possible hazards to human health through food chain. Consequently, the impacts of elevated levels of CO 2 and metal contamination on plants are receiving more attentions (Tang, 2006 2006 ). It is now known that under non-contaminated conditions, elevated levels of CO 2 increase photosynthesis, leading to the increased photosynthetic product allocation to roots. This resulted in more highly branched roots and an increase in the capacity of the root system to exploit soil volume through alteration of root morphological traits (Rogers et al., 1992 1992 ; Wechsung et al., 1999 1999 ; Prior et al., 2003 2003 ; Lee-Ho et al., 2007 2007 ). The changes in root morphology are often associated with a variation in nutrient uptake (Jia and Gray, 2007 2007 ; Jin et al., 2009 2009 ; Jin and Evans, 2010 2010 ), though considerable variation between species and systems exists (Bowes, 1993 1993 ; Kimball et al., 2002 2002 ; Franzaring et al., 2008 2008 ). Previous studies have shown the effect of elevated levels of CO 2 on plant uptakes of essential micronutrients, such as Cu, Fe, Mn, and Zn (Jia et al., 2007 2007 ; Yang et al., 2007 2007 ; Zheng et al., 2008 2008 ; Högy and Fangmeier, 2009 2009 ; Jin et al., 2009 2009 ; Li et al., 2009 2009 ), but little is known about the effect of elevated levels of CO 2 on the uptakes of non-essential elements such as Cd (Li et al., 2010 2010 ). Few studies have addressed heavy metal uptakes by plants under elevated levels of CO 2 in terms of alterations in root morphological traits. Being in direct contact with the contaminated soil or soil solution, roots may be more easily affected by changes in environmental factors, such as high cadmium (Cd) concentrations (Benavides et al., 2005 2005 ); under elevated levels of CO 2 , the root shows a greater size increase than leaves, stems, and reproductive structures, even though leaves are the main site of CO 2 exposure and uptake (Kimball et al., 2002 2002 ; Wang and Taub, 2010 2010 ). This process usually affects the efficiency of acquisition of resources (Day et al., 1996 1996 ). Cd is a non-essential element that negatively affects plant growth and development processes, such as respiration and photosynthesis (Greger and Ögren, 1991 1991 ; Baryla et al., 2001 2001 ; Vega et al., 2006 2006 ), water and mineral uptakes (Singh and Tewari, 2003 2003 ), cell division (Fojtová et al., 2002 2002 ), and cellular redox homoeostasis (Romero-Puertas et al., 2004 2004 ). From the viewpoint of water and nutrient uptakes, roots are of particular physiological importance (Bosac et al., 1995 1995 ; Jia et al., 2008 2008 ). Fine roots are usually more sensitive to exposure to either excessive metal concentrations (Arduini et al., 1995 1995 ) or elevated levels of CO 2 (Day et al., 1996 1996 ; Janssens et al., 1998 1998 ; Phillips et al., 2006 2006 ) when compared to coarse roots. Changes in root morphology may therefore serve as an important indicator of environmental changes (Nishizono et al., 1987 1987 ; Ostonen et al., 2007 2007 ). The changes in root distribution patterns may influence nutrient dynamics, thus influencing crop uptake of metals when plants are grown in contaminated soil. Although plant growth responses to elevated levels of CO 2 , when grown in metal-contaminated soil, are known (Tang et al., 2003 2003 ; Zheng et al., 2008 2008 ; Wu et al., 2009 2009 ; Li et al., 2010 2010 ), the relationship between uptakes of metals by plants exposed to elevated levels of CO 2 and metal stress and alteration of root distribution patterns remains poorly understood. As model plants, Lolium multiflorum and Lolium perenne species have been frequently studied because of their abilities to survive in metal-contaminated soil and to accumulate metals (Marseille et al., 2000 2000 ; Kiss et al., 2002 2002 ; Palazzo et al., 2003 2003 ; Arienzo et al., 2004 2004 ; Caggiano et al., 2005 2005 ; Guo and Wang, 2009 2009 ). They contain extensive root systems with high biomass, have high adaptability and low-cost management, and possess the ability to accumulate Cd (Sabreen and Sugiyama, 2008 2008 ). Understanding the combined effects of elevated levels of CO 2 and metal contamination on their biomass productions, root morphological traits, and metal accumulations will improve both our knowledge of food safety and their survival abilities in metal contaminated environments. It also allows an interspecies comparison of the behaviors of L. multiflorum vs. L. perenne in metal contaminated environments under elevated levels of CO 2 . Elevated levels of CO 2 improve photosynthesis of C 3 plants, reduce stomatal resistance, and as a result, increase water-use efficiency, while aiding in the decrease of photorespiration and oxidative stress (Urban, 2003 2003 ; Kirschbaum, 2004 2004 ; Rogers et al., 2004 2004 ). Researches have shown that elevated levels of CO 2 increase the ability of plants to combat abiotic stress, such as O 3 , drought, and salt (Sgherri et al., 1998 1998 ; Donnelly et al., 2001 2001 ; Oksanen et al., 2001 2001 ; Geissler et al., 2009 2009 ). We hypothesize that better growth and physiological responses to elevated levels of CO 2 will help plants combat the stress induced by Cd. The objective of this study was to investigate effects of elevated levels of CO 2 on plant growth, root development, and Cd uptakes of L. multiflorum and L. perenne under Cd stress, and implications for food safety and phytoremediation efficiency. Materials and methods 1 Plant materials and growth 2 Seeds of L. perenne L. and L. multiflorum Lam. (obtained from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, China) were surface-sterilized by exposure to 0.01 g/ml NaOCl for 10 min, and subsequently washed several times with deionized water, and germinated in a moist mixture of perlite and vermiculite (1:1, v/v) in a controlled growth chamber at a constant temperature (25 °C). After 10 d, healthy and uniformly-sized seedlings were selected for the hydroponic experiment. Forty-eight pots of 9.2 cm inner diameter and 18 cm height were wrapped with tinfoil, each containing 1 L of 0.50 strength Hoagland nutrient solution and one seedling. Hoagland nutrient solution (pH 6.5) consisted of 4 mmol/L Ca(NO 3 ) 2 ∙4H 2 O, 6 mmol/L KNO 3 , 2 mmol/L MgSO 4 ∙7H 2 O, 1 mmol/L NH 4 H 2 PO 4 , 15 μmol/L H 3 BO 3 , 1 μmol/L MnSO 4 ∙4H 2 O, 0.5 μmol/L ZnSO 4 ∙7H 2 O, 0.2 μmol/L CuSO 4 ∙5H 2 O, 0.01 μmol/L (NH 4 ) 6 Mo 7 O 24 , and 100 μmol/L Fe(II)-EDTA. Three days after growth in 0.50 strength nutrient solution, all pots were filled with Hoagland nutrient solution and arranged randomly into two sets. Each set, consisting of 24 pots for both species, was spiked with Cd (CdCl 2 ∙5H 2 O) at 0, 4, and 16 mg/L, with each treatment having four replicates. The nutrient solution was continuously aerated with an aquarium pump and was replaced every two days. The two sets of pots were grown in growth chambers under identical conditions varying only CO 2 level. They were grown under the following conditions: day/night time of 16/8 h, temperature of (25.0±0.5) °C, light intensity of (105±0.8) μmol/(m 2 ∙s), and a humidity of (60±1)%. Elevated levels of CO 2 were supplied by a compressed CO 2 cylinder (CO 2 purity of 99.9%) from Tianjin Saint-Nan Gases Supply Co., Ltd., China. The CO 2 concentration was monitored with an infrared gas analyzer equipped with an automatic switching solenoid used to maintain the constant CO 2 concentration of (1000±80) μl/L. The other control growth chamber was ventilated with an ambient CO 2 concentration of (360±12) μl/L. Harvest, root scanning, and chemical analyses 2 The plants were subjected to three weeks of the CO 2 treatment, and then harvested and separated into shoots and roots. Root scanning was carried out immediately using an Epson Expression 10000XL 1.0 system (Regent Instruments Company, Canada). Root length, surface area, volume, average diameter, number of tips, and root length distribution in different size-categories were measured and recorded through a root image analysis system using image analysis software WinRHIZO. The root average diameter was expressed as the total root width divided by the length of roots. After scanning, the fresh plant samples were washed with deionized water, dried in an oven at 65 °C for 72 h, and weighted and pulverized with a stainless steel cutter blender (T250D, IKA, Germany). Dry plant samples (0.200 g) were digested in 10 ml of HNO 3 -HClO 4 (4:1, v/v) at 220 °C on a hot plate, filtered with Whatman filter paper, and the filtrate was diluted to 25 ml with 5% (v/v) HNO 3 solution. The Cd concentration was determined by using an atomic absorption spectrometer (AAS) and a graphite tube equipped with an automatic sampler (ZEEnit 700, Analytik Jena, Germany). The quality control included blanks and certified samples (GBW10016) with each batch of samples. Bioconcentration factor (BCF), transport index (TI), and total Cd uptake in mass per plant 2 The Cd BCF was calculated on the basis of dry weight using Eq. ( 1 1 ). It represents an index of the ability of plants to accumulate Cd with respect to its concentration in the hydroponic substrate (Ghosh and Singh, 2005 2005 ): BCF= c Cd,sh/r ×100%/ c Cd,s , where c Cd,sh/r (mg/kg) and c Cd,s (mg/L) represent Cd concentrations in shoots or roots and the hydroponic solution, respectively. The TI was computed as the Cd concentration in plant shoots (mg/kg dry weight) divided by the Cd concentration in roots (mg/kg dry weight) using Eq. ( 2 2 ) (Ghosh and Singh, 2005 2005 ). The TI represents the ability of plants to transport Cd from root to shoot under elevated levels of CO 2 or ambient CO 2 : TI= c Cd,sh ×100%/ c Cd,r . Total shoot or root Cd uptake in mass-per-plant was calculated by shoot or root dry weight biomass multiplied by Cd concentration in the corresponding plant part. It represents the ability of plants to remove Cd from contaminated environments under elevated levels of CO 2 or ambient CO 2 . Statistical analysis 2 The experiment was performed using a three-factor completely randomized design (CRD) with four replications. Statistical analysis was performed by using SPSS statistical software (Version 16.0, SPSS Inc., Chicago, Illinois, USA). The data were analyzed using analysis of variance (ANOVA). To examine the statistical significance of differences ( P < 0.05) between means, the Tukey test was performed. Results 1 Growth of plants 2 Elevated levels of CO 2 increased the tiller number, root dry weight, shoot dry weight, and total plant dry weight significantly as compared to the ambient CO 2 controls in all the three Cd concentrations, regardless of the negative effect of Cd stress (Table 1 1 ). Toxic symptoms, as shown by brown roots and withered leaf tips, were first observed in fine root tips and leaf tips of both Lolium species at 16 mg/L Cd exposure. The plants grown under elevated levels of CO 2 showed less Cd toxicity symptoms (data not shown), implying that increased root growth due to elevated levels of CO 2 may alleviate Cd toxicity. Cd exposure increased dry weight ratios of root/shoot in both plant species, but the elevated levels of CO 2 had the opposite effect (Table 1 1 ). Apparently, there were interacting effects between Cd and CO 2 on the shoot dry weight, root dry weight, and total plant dry weight for both plant species ( P < 0.05; Table 2 2 ). Significant differences in tiller numbers and root/shoot ratios were noted between the two plant species in terms of the response to elevated levels of CO 2 ( P < 0.05). c Cd : concentration of Cd; n : number of tillers; DW: dry weight; TPDW: total plant dry weight. Values are means of four replicates. For each species, different small superscript letters or capital superscript letters following values in the same column refer to significant differences ( P < 0.05) between Cd treatments at ambient or elevated CO 2 levels, respectively Within a Cd concentration, ‘*’ indicates significant differences ( P < 0.05) between the two CO 2 treatments in each species Species CO 2 level c Cd (mg/L) n Shoot DW (g) Root DW (g) TPDW (g) Root/shoot ratio L. multiflorum Ambient CO 2 0 18.0 a 1.04 a 0.249 a 1.29 a 0.244 b 4 15.3 b 0.53 b 0.223 a 0.75 b 0.409 a 16 10.5 c 0.40 c 0.165 b 0.57 c 0.418 a Elevated CO 2 0 20.5 A 1.75 A * * 0.411 A * * 2.16 A * * 0.234 B 4 15.8 B 1.02 B * * 0.344 B * * 1.36 B * * 0.338 A * * 16 11.8 C 0.76 C * * 0.249 C * * 1.01 C * * 0.346 A * * 8 L. perenne Ambient CO 2 0 14.8 a 1.11 a 0.249 a 1.36 a 0.225 b 4 11.3 b 0.54 b 0.186 b 0.73 b 0.327 a 16 7.5 c 0.42 b 0.148 c 0.57 c 0.348 a Elevated CO 2 0 23.5 A * * 1.83 A * * 0.366 A * * 2.20 A * * 0.200 B 4 17.8 B * * 0.87 B * * 0.310 B * * 1.23 B * * 0.302 A 16 12.3 C * * 0.70 B * * 0.236 C * * 0.94 C * * 0.321 A n : number of tillers; DW: dry weight; TPDW: total plant dry weight. n.s.: no significant differences P < 0.05 P < 0.01 P < 0.001 2 Factor 5 F value n Shoot DW Root DW TPDW Root/shoot ratio Species 5.2 * * n.s 5.7 * * n.s 5.0 * * CO 2 81.0 *** *** 257.2 *** *** 193.0 *** *** 323.7 *** *** 6.6 * * Cd 116.3 *** *** 4.5 *** *** 90.8 *** *** 294.7 *** *** 39.3 *** *** Species×CO 2 37.2 *** *** n.s. n.s. n.s. 11.6 ** ** Species×Cd n.s. n.s. n.s. n.s. n.s. CO 2 ×Cd n.s. 37.6 *** *** 11.4 *** *** 37.7 *** *** n.s. Species×CO 2 ×Cd n.s. n.s. n.s. n.s. n.s. Elevated levels of CO 2 triggered an increase in total plant dry weight (Table 1 1 ). The increased stimulation due to elevated levels of CO 2 was 67%, 77%, and 81%, for L. multiflorum , and 62%, 65%, and 68% for L . perenne under 0, 4, and 16 mg/L Cd treatments, respectively. The increased shoot biomass was likely dependent on the increase in tiller number for L . perenne , and more relied on leaf expansion for L. multiflorum (data not shown). The elevated CO 2 treatment showed more dry weight increase in shoots than in roots, resulting in reduced root/shoot ratio compared to the ambient CO 2 control. Root morphological traits 2 It was clear that for the same Cd level, elevated levels of CO 2 improved the root growth of the two tested species (Tables 3 3 and 4 4 ). Elevated levels of CO 2 increased the root length by 8.6%, 17.0%, and 14.0% for L. multiflorum , and by 35.8%, 42.9%, and 46.9% for L . perenne under 0, 4, and 16 mg/L Cd treatments, respectively; it also increased the root tip numbers by 0.7%, 8.8%, and 7.9% for L. multiflorum , and by 4.8%, 39.4%, and 47.7% for L . perenne under 0, 4, and 16 mg/L Cd treatments, respectively. When both plant species were exposed to Cd under elevated levels of CO 2 , the negative effect of Cd on the roots of both plant species was mitigated and consequently roots with a larger volume were observed under the elevated CO 2 condition compared to the ambient CO 2 control (Tables 3 3 and 4 4 ). The elevated CO 2 treatment increased the average root diameter of L. multiflorum , but decreased average root diameter of L. perenne . c Cd : concentration of Cd; l : length; S : surface area; V : volume; d : average diameter; n tips : number of tips. Values are means of four replicates. For each species, different small superscript letters or capital superscript letters following values in the same column refer to significant differences ( P < 0.05) between Cd treatments at ambient or elevated CO 2 levles, respectively. Within a Cd concentration, ‘*’ indicates significant differences ( P < 0.05) between the two CO 2 treatments in each species Species CO 2 level c Cd (mg/L) l (cm) S (cm 2 ) V (cm 3 ) d (mm) n tips L. multiflorum Ambient CO 2 0 185 a 299 a 3.86 a 0.516 b 2519 a 4 133 b 216 b 2.83 b 0.511 b 2044 b 16 93 c 161 c 1.73 c 0.549 a 1805 b Elevated CO 2 0 201 A 359 A * * 5.91 A * * 0.637 AB * * 2539 A 4 156 B 296 B * * 4.55 B * * 0.610 B * * 2225 AB 16 106 C 179 C 2.77 C * * 0.647 A * * 1947 B 8 L. perenne Ambient CO 2 0 201 a 304 a 3.67 a 0.582 b 4467 a 4 84 b 145 b 2.03 b 0.565 b 1466 b 16 49 c 87 c 1.21 c 0.677 a 1016 c Elevated CO 2 0 273 A * * 549 A * * 5.82 A * * 0.544 AB * * 4680 A 4 120 B * * 178 B * * 3.23 B * * 0.507 B * * 2044 B * * 16 72 C * * 13 C * * 2.06 C * * 0.586 A * * 1501 C * * l : length; S : surface area; V : volume; d : average diameter; n tips : number of tips. n.s.: no significant differences P < 0.05 P < 0.01 P < 0.001 2 Factor 5 F value l S V d n tips Species 11.8 ** ** n.s. n.s. n.s. n.s. CO 2 37.7 *** *** 29.0 *** *** 141.6 *** *** 4.3 * * n.s. Cd 227.3 *** *** 79.0 *** *** 155.4 *** *** n.s. 71.8 *** *** Species×CO 2 9.2 ** ** 4.9 * * 12.2 ** ** n.s. 5.6 * * Species×Cd 33.1 *** *** 14.7 *** *** 23.5 *** *** n.s. 35.4 *** *** CO 2 ×Cd n.s. 3.7 * * 22.0 *** *** 4.2 * * n.s. Species×CO 2 ×Cd n.s. 4.3 * * 9.5 ** ** n.s. n.s. Increasing Cd concentrations resulted in a significant decrease in number and proportion of fine roots in both species (Fig. 1 1 ). Elevated levels of CO 2 alleviated the decreases of fine roots in both Cd-stressed species, but the percentage of thick roots showed a positive response to elevated levels of CO 2 for L. multiflorum and a negative response for L. perenne . Dameter: fine, < 0.1 mm; medium, 0.1–1.0 mm; thick, > 1.0 mm. For each species, different letters refer to significant differences ( P < 0.05) between Cd treatments at ambient or elevated CO 2 levels for each size-category, respectively. Within a Cd concentration, ‘ * ’ indicate significant differences ( P < 0.05) between the two CO 2 treatments in each size-category for each species Cd concentration and total uptake 2 Plants grown under elevated levels of CO 2 had lower Cd in both shoots and roots, compared to the ambient CO 2 control. The effect of interaction between Cd and CO 2 on Cd concentration in shoots and roots was significant ( P < 0.05). The decrease of Cd concentration in shoots and roots triggered by elevated levels of CO 2 was more substantial in 16 mg/kg Cd treatment than in 4 mg/kg Cd treatment (Fig. 2 2 ). However, the total Cd uptake calculated on the basis of per-pot dry weight biomass was significantly higher (42% to 73% increase in shoots) at elevated levels of CO 2 than at ambient CO 2 levels (Fig. 3 3 ). For shoot or root of each species: different letters refer to significant differences ( P < 0.05) between treatments; n.d.: not determined. For ANOVA: n.s.: no significant differences; * P < 0.05, ** P < 0.01, *** P < 0.001 For shoot or root of each species: different letters refer to significant differences ( P < 0.05) between treatments; n.d.: not determined. For ANOVA: n.s.: no significant differences; * P < 0.05, ** P < 0.01, *** P < 0.001 At ambient levels of CO 2 and high Cd levels (16 mg/L), the measured levels of Cd were 417 and 466 mg/L in the shoots, and 3 469 and 4 460 mg/L in the roots of L. multiflorum and L. perenne , respectively (Fig. 2 2 ). The BCF of root was much higher than that of shoot (Tables 5 5 and 6 6 ). Both BCFs decreased with increasing spiked Cd concentration in the growth media regardless of CO 2 treatment. At a lower Cd concentration (4 mg/L) and ambient CO 2 levels, the BCFs of roots and shoots were as high as 388 and 39 L/kg for L. multiflorum , and 436 and 39 L/kg for L. perenne , respectively. However, the BCFs of roots and shoots decreased with elevated levels of CO 2 regardless of Cd levels. TI values throughout the treatments were less than 1, and lower for the 16 mg/L Cd treatment than for the 4 mg/L Cd treatment. Elevated levels of CO 2 induced a higher TI in both species under the same Cd treatment level. LM: L. multiflorum ; LP: L. perenne ; c Cd : concentration of Cd; BCF: bioconcentration factor; TI: transport index. Values are means of four replicates. For each species, different small superscript letters or capital superscript letters following values in the same column refer to significant differences ( P < 0.05) between Cd treatments at ambient or elevated CO 2 , respectively Within a Cd concentration, ‘*’ indicates significant differences ( P < 0.05) between the two CO 2 treatments in each species 2 Species 2 CO 2 level 2 c Cd (mg/L) 2 BCF (L/kg) 2 TI Shoot Root LM Ambient CO 2 4 39 a 388 a 0.100 a 16 26 b 217 b 0.088 b Elevated CO 2 4 35 A 337 A 0.105 A * * 16 23 B * * 167 B * * 0.092 B 6 LP Ambient CO 2 4 39 a 436 a 0.120 a 16 29 b 279 b 0.105 b Elevated CO 2 4 37 A 404 A 0.139 A * * 16 24 B * * 207 B * * 0.117 B * * BCF: bioconcentration factor; TI: transport index; n.s.: no significant differences P < 0.05 P < 0.01 P < 0.001 3 Factor 3 F value 2 BCF (L/kg) 2 TI Shoot Root Species n.s. 18.02 *** *** 9.93 ** ** CO 2 5.02 * * 15.91 ** ** 4.62 * * Cd 57.96 *** *** 183.43 *** *** 21.75 *** *** Species×CO 2 n.s. n.s. n.s. Species×Cd n.s. n.s. n.s. CO 2 ×Cd n.s. n.s. n.s. Species×CO 2 ×Cd n.s. n.s. n.s. Discussion 1 Combined effects of Cd concentrations and elevated CO 2 levels on plant growth 2 We observed a significant effect of elevated levels of CO 2 on the dry weights of both Lolium plants ( P < 0.001) (Tables 1 1 and 2 2 ); however interestingly, the elevated levels of CO 2 had an increasing effect on the total plant dry weight but it was different for different plant species under 0, 4, and 16 mg/L Cd treatments. Enhanced plant growth under elevated levels of CO 2 and uncontaminated soil conditions has been widely documented, and the magnitude of increase was dependent on plant species and genotype (Ziska et al., 1996 1996 ; Moya et al., 1998 1998 ; Horie et al., 2000 2000 ; Kimball et al., 2002 2002 ; Long et al., 2004 2004 ; Lobell and Field, 2008 2008 ; Cheng et al., 2009 2009 ). The increased stimulation, due to elevated levels of CO 2 , was higher under Cd stress than under non-Cd stress for both species, showing the effectiveness of elevated levels of CO 2 in ameliorating Cd toxicity. This may be partially related to the root development and alteration of the root morphological traits (Peng et al., 2005 2005 ). The root/shoot ratio response to elevated levels of CO 2 differed among species and growth conditions (Ferris and Taylor, 1993 1993 ). From the viewpoint of phytoextraction, our finding that CO 2 level elevation increased the biomass of the two Lolium species grown in hydroponic solutions spiked with various levels of Cd, may also have implications for improvement of phytoextraction efficiency as well. When compared the two Lolium species, greater reduction in plant growth, due to spiked Cd in growth media, was observed in L. perenne than in L. multiflorum , suggesting that the latter has higher metal tolerance than the former. Similar reports were documented in Sabreen and Sugiyama ( 2008 2008 ) who showed that L. perenne had a higher growth inhibition than L. multiflorum under Cd stress. Combined effect of Cd concentrations and elevated CO 2 levels on root morphological traits 2 Root length, surface area, volume, and tip number are important parameters for understanding how root systems respond to environmental changes, such as increases in CO 2 levels and Cd stress. Both Lolium species had higher values of the root morphological parameters (including root length, surface area, volume, and tip numbers) at elevated levels of CO 2 when compared to the ambient CO 2 control, showing that elevated levels of CO 2 increased root elongation and root branching. Corresponding to the response of plant growth to elevated levels of CO 2 , Cd treatments affected root tip number, root length, surface area, volume, and number of fine roots as reported in Daud et al. ( 2009 2009 ) and Li et al. ( 2009 2009 ). Generally, root length, surface area, and volume are more sensitive to Cd than root tip number (Ci et al., 2009 2009 ). Fine roots were more affected than the coarse ones when exposed to Cd (Cosio et al., 2006 2006 ). The exposure of plants to Cd stress reduced the nutrition uptake from growth media by affecting root growth, elongation, and absorption zones (Peng et al., 2005 2005 ), and thus inhibited the growth of plants. Our results showed that L . perenne was more sensitive to Cd than L . multiflorum as shown by their differences in the root parameters (Tables 3 3 and 4 4 ). For L. multiflorum , the root tip number was less influenced than the root length, surface area, and volume. A more substantial decrease of root tip number was observed in L. perenne than in L. multiflorum . Since the root tip number partly reflects the lateral root emergence, the higher inhibition of lateral root emergence in L. perenne may be used to explain why it had more inhibited root growth than L. multiflorum . For both Lolium species, we observed a more substantial decrease in fine roots than coarse roots although the latter was also strongly inhibited. For both Lolium species, elevated levels of CO 2 triggered a more substantial increase in root length, volume, and tip number under Cd stress than under non-Cd treatments when compared to the ambient CO 2 control. Elevated levels of CO 2 caused a significant increase of root length in all three root size-categories, but the magnitude of increase varied to different degrees. It is widely documented that elevated levels of CO 2 increased fine root numbers (Pritchard and Rogers, 1999 1999 ; Matamala and Schlesinger, 2000 2000 ; Pritchard et al., 2001 2001 ). Our study showed that the fine root proportion was not affected by elevated levels of CO 2 under the Cd control treatment, but it increased when plants were exposed to Cd. This suggested that under the Cd stress condition, elevated levels of CO 2 stimulated more fine roots than coarse ones, indicating that alleviation of Cd toxicity in response to elevated levels of CO 2 may be related to an increase in fine root numbers. The increased fine roots aid the nutrient and water uptake as they are the most active parts of the roots. This can explain why elevated levels of CO 2 triggered more increase of plant biomass under Cd stress than under non-Cd treatment compared to the ambient CO 2 control. It was more likely that plants obtained additional energy from the photosynthesis to combat with the stress under elevated levels of CO 2 by altering root morphological traits (Tables 1 1 and 2 2 ). Effects of elevated CO 2 levels on Cd uptake and implication for food safety and phytoremediation 2 Both of the two Lolium species showed potential materials for phytoremediation due to their high Cd uptakes. Our study showed a reduction of Cd concentration in the two tested plant species by the lower BCF, especially in the roots, at elevated levels of CO 2 . The lower Cd concentration under elevated levels of CO 2 will benefit plants grown under Cd stress. This reduction in Cd concentration might be related to the so-called dilution effect induced by fast plant growth as reported in Loladze ( 2002 2002 ). Lieffering et al. ( 2004 2004 ) hypothesized that if the root production response under elevated levels of CO 2 was much higher than the above-ground biomass response, the elemental uptake may match the increase in above-ground biomass. In our case, the relatively lower biomass increase in roots of the two Lolium species under elevated levels of CO 2 (as showed by root/shoot ratio) may suggest a Cd dilution mechanism. Further research is required to determine whether elevated levels of CO 2 interact with the Cd influence on membrane permeability and the activity of the transport protein, thus altering the allocation of elements within the plant. Despite a reduction of Cd concentration in plant species at elevated levels of CO 2 , we observed higher TI at elevated levels of CO 2 than at ambient CO 2 , suggesting that elevated levels of CO 2 improved Cd transportation from roots to shoots (Tables 5 5 and 6 6 ). The increased TI may be due to the lower Cd concentration in plants under elevated levels of CO 2 , as the higher TI was observed in lower Cd stress. Taking into consideration the reduction of Cd concentration in plant tissues under elevated levels of CO 2 , we speculate that the improvement in Cd transportation from roots to shoots may be insufficient to compensate the dilution effect due to increased biomass under elevated levels of CO 2 . A survey of literature shows that dilution phenomena are well documented for some staple food crops in the pot studies where nutrient supplies are generally limited. Högy and Fangmeier ( 2009 2009 ) observed a 3.7%–18.3% reduction in all micro-elements in wheat grown in the different exposure systems. Guo et al. ( 2006 2006 ) reported decreased Cd accumulation in leaves, stems, roots and grains of rice at elevated CO 2 . Zheng et al. ( 2008 2008 ) showed that Pteridium revolutum and Pteridium aquilinum grown on Cu-contaminated soils accumulated less Cu in plant tissues at elevated levels of CO 2 than at ambient CO 2 . Li et al. ( 2010 2010 ) found that elevated levels of CO 2 diluted grain Cd concentration. In view of the potential benefits of CO 2 -triggered “metal dilution” and the very limited available data on the subject, we speculate that this might have an important positive implication for the food safety regarding the contaminated soil from which crops are harvested. Calculation of total Cd uptake in each plant tissue from each pot showed that there was a much higher total Cd uptake at elevated levels of CO 2 than at the ambient CO 2 control (an increase of 42.2% to 73.4% for shoots). Taking into account our present results and previously obtained results (Wu et al., 2009 2009 ; Jia et al., 2010 2010 ; Li et al., 2010 2010 ), we proposed that enriching CO 2 in growth chambers can help plants remove more metals from growth media as indicated by their enhanced metal total uptake. This might have positive implications for improving phytoremediation efficiency if enriching CO 2 in growth chambers is used to assist phytoextraction of the contaminated soil. Conclusions 1 Root morphological parameters, including root length, surface area, volume, tip number, and fine roots, all decreased under Cd exposure. By contrast, elevated levels of CO 2 significantly increased all those parameters in the presence of Cd, compared to the CO 2 control, suggesting that elevated levels of CO 2 had an ameliorating effect on Cd-induced stress. The significantly higher total Cd uptake per pot, calculated on the basis of biomass under elevated levels of CO 2 in association with increased biomass production, may have implications for food safety and phytoextraction. 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DOI: 10.1080/15226510801913934 http://doi.org/10.1080/15226510801913934 . Using elevated carbon dioxide to enhance copper accumulation in Pteridium Revolutum, a copper-tolerant plant, under experimental conditions LH Ziska , PA Manalo , RA Ordonez . Intraspecific variation in the response of rice ( Oryza sativa L.) to increased CO 2 and temperature-growth and yield response of 17 cultivars . J Exp Bot , 1996 . 47 ( 9 ): 1353 - 1359 . DOI: 10.1093/jxb/47.9.1353 http://doi.org/10.1093/jxb/47.9.1353 . Intraspecific variation in the response of rice (Oryza sativa L.) to increased CO2 and temperature-growth and yield response of 17 cultivars We want to thank Dr. Lena Q. MA (University of Florida, USA) and Dr. Hadrian F. COOK (Department of Agricultural Sciences, Imperial College, Wye, Kent, UK) for critically reviewing the manuscript. 10.1631/jzus.B1000181 Project supported by the Central Public Research Institute Basic Fund for Research and Development (2008-jxh-1), Agro-environmental Protection Institute, Ministry of Agriculture, China 2010-05-17 2011-03-02 2010-11-15 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2011 2011 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 04 12