Figure 4

Functional analysis of a tolerance-associated gene encoding LRR-RLK VIII in response to As stress. (a) Identification of T-DNA insertion for LRR-RLK VIII mutations in the Arabidopsis genome by PCR Analysis. The left (LB) border primers for T-DNA insertion, and the left (LP) and right (RP) genomic primers for LRR-RLK VIII genes are presented in materials and methods. The sequences of junctions between T-DNA borders and the genomic target were detected in homologous LRR-RLK VIII mutants when compared to the wild-type plants. Two T-DNA mutant lines for locus AT1G53440 were characterized, and one for AT1G53430 (upper panel). The gene expression of LRR-RLK VIII (AT1G53440) in the mutant line (Salk_057812) was analyzed by RT-PCR (bottom panel). There was no characterized T-DNA mutant line for AT3G09010. (b) Effect of As (V) on the root elongation of Arabidopsis wild-type and LRR-RLK VIII mutant lines was assessed. Measurement of root elongation was similar to that described in Figure 1. Seedlings were grown on quarter-strength MS medium for 4 d and then transferred to medium with 100 and 200 μM As (V) and grown for an additional 4 d. As tolerance was determined by relative root growth after treatment. Root length of plants was measured after treatment with As. Root samples were collected from 3 independent experiments (each from a pool of 7 root samples). Data are mean±SD. *P ≤ 0.05 compared to As treatment (200 μM) from wild-type plants. The difference in root elongation is significant according to Student's t test. (c) Accumulation of As (V) by Arabidopsis wild-type and LRR-RLK VIII mutant lines was analyzed with the methods similar to Figure 1. Data are mean±SD calculated from 3 biological replicates per treatment. *P ≤ 0.05 compared with wild-type plants. (d) Effect of As (200 μM), Cu (50 μM) and H₂O₂ (100 μM) on the root elongation of Arabidopsis wild-type and LRR-RLK VIII mutant lines. Measurement of root elongation was similar to that described in Figure 1. At least three biological replicates were performed corresponding to each treatment. Data are the mean ± SE of three independent replicates.