Electronic Supplementary Material for
Physiological and biochemical response of soil-grown barley (Hordeum vulgare L.) to cerium oxide nanoparticles
Cyren M. Rico,1,2 Ana C. Barrios,1 Wenjuan Tan,1 Rosnah Rubenecia,3 Sang Chul Lee,3 Armando Varela-Ramirez,4 Jose R. Peralta-Videa,1,2,5 and Jorge L. Gardea-Torresdey*1,2,5
1Department of Chemistry, The University of Texas at El Paso, 500 W. University Avenue, El Paso Texas 79968, United States
2University of California Center for Environmental Implications of Nanotechnology (UC CEIN), The University of Texas at El Paso
3School of Applied Biosciences, Kyungpook National University, Daegu 702-701, Republic of Korea
4Department of Biological Sciences, Border Biomedical Research Center, The University of Texas at El Paso, 500 W. University Avenue, El Paso Texas 79968, United States
5Environmental Science and Engineering PhD Program, The University of Texas at El Paso, 500 W. University Avenue, El Paso Texas 79968, United States
*Corresponding author. Tel: 915 747-5359; fax: 915 747 5748. E-mail address: (J. Gardea-Torresdey)
Journal: Environmental Science and Pollution Research
Prepared: February 3, 2015
6 pages in length, including 3 tables
Measurement of membrane damage
Membrane damage was determined by measuring the leakages of K and UV absorbing substances from the cells (K leakage and UVAS ratio, respectively). The measurement of K leakage was performed according to Navarri-Izzo et al. (1989). The leaves were cut into small pieces, washed three times, placed in 15 mL MW, and incubated with shaking at RT for 4 h. The supernatant was collected and analyzed for K content by ICP-OES. UVAS ratio was estimated following the method of Lutts et al. (1996) The samples were incubated in 10 mL MW for 24 h and an aliquot was used to read the absorbance at 280 nm. The aliquot was added back to the original solution before freezing the solution for 6 h. The absorbance was measured again and the UVAS was calculated by taking the ratio between the initial and final absorbance reading.
Cerium, macro, and micronutrient concentrations in barley
Quantitative analyses of elemental concentrations in barley tissues were performed according to the method previously employed by Rico et al. (2014). The samples were digested in a mixture of plasma pure HNO3 and H2O2 (1:4) using a microwave accelerated reaction system (CEM MarsX Mathews, NC). ICP-OES/MS (Perkin Elmer, Waltham, MA) were used for the analyses. Blank, spikes and standard reference material (NIST-SRF 1570a) were used to validate the digestion and analytical method.
Amino acid profiling
The method described by Anjum et al. (2005) was employed to analysis of amino acid contents in barley grains. Briefly, a 5 mL 6 N HCl was used to homogenized the powdered grains (25 mg) in a test tube which was subsequently evacuated by nitrogen, sealed, and further incubated in an oven (110°C) for 24 h. The solution was filtered and dried under vacuum (60°C) before dissolving in 1 mL phosphate buffer (pH 2.2). An aliquot (100 µL) was diluted 10 times with buffer, filtered in a 45 µm syringe filter, and analyzed using Biochrom 20 amino acid analyzer (Biochrom, USA).
Fatty acid analysis
Fatty acid content in barley grains was estimated according to Asekova et al. (2014). The powdered grains (500 mg) were digested in a 5 mL solution of chloroform:hexane:methanol (8:5:2 v/v/v) overnight. A 100-µL extract was obtained and mixed with 75 µL of methylating reagent (0.25 M methanolic sodiummethoxide: petroleum ether: ethyl ether [1:5:2, v/v/v]) for 10 h before diluting the solution to 1 mL using hexane. Fatty acid content was analyzed using an Agilent (Palo Alto, CA) Series 7890 capillary gas chromatograph fitted with a flame ionization detector with an AT-Silar capillary column (Alltech Associates, Derfield, IL). The temperatures of the oven, injector, and detector were set at 210, 250, and 230°C, respectively. Standard fatty acid mixtures (Animal and Vegetable Oil Reference Mixture 6, AOACS) were used as calibration reference standards.
SI Table 1. Fertilization record of barley cultivated to grain production in nCeO2-amended soil.
0 / 200
1 / 200
26 / 200
122 / 300
SI Table 2. Yield characters of barley cultivated to grain production in nCeO2-amended soil. Values are means ± SE (n = 3). Same letters mean no statistical difference between treatments at Tukey’s test (p £ 0.05).
(mg kg-1) / yield components
number of grains per spike / grain yield per spike
(mg) / yield per pot
(g)
0 / 6.3 ± 0.9a / 153.0 ± 18.6a / 0.99 ± 0.1a
125 / 6.4 ± 0.9a / 179.4 ± 19.9a / 1.18 ± 0.3a
250 / 7.6 ± 0.9a / 212.6 ± 30.4a / 1.56 ± 0.4a
SI Table 3. Soluble protein content in leaves of wheat cultivated in nCeO2-amended soil at 48 days after transplanting. Values are means ± SE (n = 3). Same letters in each column mean no statistical difference between treatments at Tukey’s test (p £ 0.05).
nCeO2 concentration(mg kg-1) / soluble protein (mg g-1)
0 / 3.87 ± 0.37b
125 / 4.86 ± 0.37ab
250 / 5.74 ± 0.63ab
500 / 26.81 ± 0.66a
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