Three plant species were used in the present study: rice (O. sativa L.), because of the frequent detection of methylated As in rice grains; tomato (S. lycopersicum L.), because of the findings of Nissen & Benson (1982); and red clover (Trifolium pratense L.), because high proportions of MMA(V), accounting for 57% of total As, were reported in shoot samples collected near an arsenopyrite ore vein (Geiszinger et al., 2002). Three rice cultivars were used: two japonica cultivars, Nipponbare and Italica carolina, and one indica cultivar, Kasalath. Rice seeds were dehusked and sterilized by washing twice with 70% ethanol, followed by 30 min in 1% active NaOCl. Seeds were washed thoroughly with sterile water and left to germinate in 1% Plant Preservative Mixture™ solution (Plant Cell Technology, Washington, DC, USA) for 5–7 d in a growth cabinet at 27°C day : 20°C night temperatures and a 14-h photoperiod with a light intensity of 300 μmol m−2 s−1. After germination, four seedlings were placed on full-strength Yoshida nutrient solution (Yoshida et al., 1976) set with 1% agar in coupled Magenta™ vessels (Sigma, St Louis, MO, USA) and were grown under the same conditions until harvest. Tomato (cv Alicante) seeds were sterilized in 1% active NaOCl for 10 min and washed thoroughly with sterile water, and four seeds were placed on half-strength Murashige and Skoog (MS) growth medium (Murashige & Skoog, 1962) set with 1% agar in each Phytatray™ II box (Sigma). Plants were grown at a constant temperature of 22°C and a 16-h photoperiod with a light intensity of 250 μmol m−2 s−1. The experiment with red clover was conducted using a modified most probable number method (Vincent, 1970). Briefly, red clover seeds were sterilized in 95% H2SO4 for 5 min, rinsed thoroughly with sterile water and germinated on 1% agar for 3 d. One germinated seedling was placed on each slope of sterile quarter-strength Hewitt’s nutrient solution (Hewitt, 1966), lacking major nitrogen sources, set with 1.5% agar in boiling tubes. After 1 wk of growth, 100 μl of Rhizobium leguminosarum (bv. trifolii) cultured in yeast extract-mannitol broth (YM) was placed on each plant’s root system. Control plants were grown on medium amended with 0.26 mM NH4NO3 and were inoculated with 100 μl of sterile YM. Plants were grown under the same conditions as in the tomato experiment. Before harvest, nodule number was recorded and four to five plants with similar numbers of nodules were grouped together to form a single replicate. Arsenic was supplied as arsenate (10 μM) for all three plant species, and additionally as arsenite (10 μM) or 5 μM MMA(V) or DMA(V) for rice. For rice and tomato, a number of secondary treatments were imposed, namely control (complete nutrients), low nitrogen (N), low phosphorus (P), and low N and P (low NP). For rice, concentrations of N and P were decreased from 1.4 and 0.32 mM in the control Yoshida medium to 200 and 10 μM, respectively. For tomato the concentrations of N and P were also reduced to 200 and 10 μM from 30.0 and 0.62 mM, respectively, in the control half-strength MS.
After sufficient growth (30–38 d) plants were removed from agar, separated into roots and shoots and weighed. Shoots and roots were rinsed with deionized water, and submerged in ice-cold desorption solution (1 mM K2HPO4, 0.5 mM Ca(NO3)2 and 5 mM MES, pH 6.0) for 15 min with periodic shaking to remove apoplastic As. Samples were ground in liquid nitrogen using a pestle and mortar. Phosphate buffer solution (PBS; 2 mM NaH2PO4 and 0.2 mM Na2-EDTA, pH 6.0) was added to the finely ground shoot and root samples, sonicated for 1 h and then double-filtered through Whatman 40 filter papers and 0.2-μm filters for As speciation analysis by HPLC-ICP-MS.