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One hundred segments for each measurement were collected into a 10?mL plastic centrifuge tube containing 8?mL of 0.5?mm CaCl2 solution at pH 4.5 (control solution) with three replicates. The plastic centrifuge tube was placed on a shaker (20?r.p.m.) for 30?min. The root segments were washed with 8?mL of the same solution three times Furin to remove any oxalic acid released from the wounded tissue and then transferred into a new 10?mL plastic centrifuge tube. Treatment was initiated by replacing the solution with 8?mL of 0.5?mm CaCl2 solution containing 0 or 10??m Cd, pH 4.5, and the tube was placed on an orbital shaker (20?r.p.m.). After 6?h, the root exudates were collected. Endogenous soluble oxalic acid in different sections of the roots was extracted three times with distilled water at 65?��C. The extracts were applied to cation- and anion-exchange resins and analysed as described previously. To examine the effect of three anion-channel inhibitors on Cd-induced secretion of oxalic acid, roots were treated with CdCl2 (10??m) in 0.5?mm CaCl2 containing 10??m niflumic acid (NIF, Sigma, St Louis, MO, USA) or 10??m anthracene-9-carboxylic acid (A-9-C, Sigma-Aldrich, Steinheim, Germany) dissolved in ethanol or 10??m phenylglyoxal (PG, Katayama Chemical, Osaka, Japan) dissolved in distilled water. Root Trametinib order exudates were collected for 4?h after the treatment. In some cases, plants were harvested for Cd content determination. The toxicity of Cd-oxalate complexes to plant root was assayed by exposing buckwheat root to different ratios of Cd to oxalate. Seeds were soaked in water for 10?h and then germinated on moist filter paper in an incubator at 30?��C. After 1?d, the seedlings were transferred into a net tray containing 0.5?mm CaCl2 solution at pH 4.5 in a growth chamber under the following conditions: 25?��C day and 20?��C night, 65% RH, light intensity 400??mol Selleckchem Enzalutamide photon m?2?s?1, and a 14-h photoperiod. After a further 2?d, seedlings of similar size were selected and subjected to the following treatments in 0.5?mm CaCl2 solution at pH 4.5 (six replicates): (1) ?Cd (control, no Cd addition); (2) +Cd (10??m as CdCl2); (c) Cd-oxalate at a 1:1 molar ratio; (d) Cd-oxalate at a 1:2 molar ratio. Different Cd-oxalate complexes were prepared by mixing CdCl2 and oxalic acid at different molar ratios. Root length was measured with a ruler before and after treatment (24?h), and seedlings were harvested and separated into roots and shoots for metal content analysis. Each experiment was repeated at least three times. Data were analysed by one-way analysis of variance procedure and the means were compared by Duncan's multiple range test. Different letters on the histograms indicate that the means were statistically different at P?
