74 H. Guo et al.
The rice and wheat plants from the first sowing were sampled at the mid-tiller-
ing, panicle-initiation, and grain-maturity growth stages, and those from the second
sowing were only sampled at grain maturity. Leaves for enzyme assays were frozen
in liquid nitrogen when sampling and stored at −80 °C. Soils were sampled at the
rice grain-maturity growth stage of the second sowing (October 2007) for pH analy-
sis and metal fractionations. Rice roots were sampled at the panicle-initiation stage
of the third sowing and stored at − 40 °C for microscopy (August 2008).
4.2.4 Analysis of Antioxidant Enzyme Activities in Leaves
The preparation method for crude enzyme referred to the method proposed by Cho
and Seo ( 2005 ). The activities of superoxide dismutase (SOD) were measured by
nitroblue tetrazolium (NBT) photoreduction method (Dhindsa et al. 1981 ). The
methods for determining the activities of catalase (CAT), peroxidase (POD), and
ascorbate peroxidase (APX) were as described earlier (Cakmak and Horst 1991 ;
Cakmak et al. 1993 ; Asada 1984 ).
4.2.5 Determination of Cd and Cu
The sampled plants were thoroughly washed with tap water and then with deionized
water, and were then oven-dried to a constant weight at 70 °C. The dried samples
were ground, weighed, and digested with concentrated HNO 3 /HClO 4 (4:1 v/v; Li
et al. 2001 ). The Cd and Cu concentrations in the digested solution were analyzed
by atomic absorption spectroscopy (AAS; Thermo Solaar M6, USA).
4.2.6 Determination of Soil pH and Sequential Extraction of Soil
The pH of the soil was measured in a 0.01 M CaCl 2 solution at a 1:2.5 ratio of soil
to solution (w/v) using a pH meter. Cu and Cd fractionation in soil was determined
by sequential extraction using the method of the Commission of the European Com-
munities (Community Bureau of Reference; BCR). The method is described in de-
tail by Quevauviller et al. ( 1997 ).
4.2.7 Scanning Electron Microscopy
Fresh rice roots were thoroughly washed with deionized water. The first 1 cm of
each root tip was cut and coated with gold (ca. 1 nm thickness) for 60 s using a
sputter coater (HITACHI E-1010, Japan). The samples were viewed with a scanning
electron microscope (SEM; S-3400N II, Hitachi, Japan).