Isolation and characterization of aluminum stress resistant genes using activati on tagging lines of Arabidopsis thaliana

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To isolate new Al resistant genes, we performed a screening of resistant plants from a pool of Arabidopsis enhancer tagging lines. From approximately 16,000 lines, 20 clones were isolated as Al resistant candidates. Southern hybridization indicated that one line, #355-2, only carries single copy of the enhancer tag in chromosome DNA. A TAIL-PCR was performed for #355-2, and the result indicated that the tag is inserted in chromosome I (very closed to up-stream region of the F9E10.5 gene). RT-PCR was also carried out and we found that a higher gene expression of F9E10.5 and F9E10.6 was specifically detected in #355-2 line (approximately 8-and 7-folds, respectively) than control line (Col-0). DNA database analysts indicated that F9E10.6 encodes a DNA glycosylase, but the function of F9E10.5 is unknown.To characterize the A resistant mechanisms in #355-2 line, morphological observation of Col-0 and #355-2 was performed and we could see a clear difference in root-hair region. The length of root-hair of #355-2 was much shorter (approximately 1/3) than that of Col-o. Al treatment (300-500 μM, 2d) for these two lines indicated that their root-hairs were completely damaged by Al toxicity. Moreover, accumulation of Al in root region of #355-2 was smaller than that of Col-0. From these results, we speculated that root hair is one of the targets for Al attack and that the lower accumulation of Al ions in root region may be one of the Al resistant mechanisms in #355-2. These results also suggested that the Al influx is probably occurred not only in root-tip, but also in root-hair.We constructed two types of transgenic lines, over-expressing and suppressing of the F9E10.5 gene and determined their Al sensitivity and root-hair formation, but we suggested that F9E10.5 may not relate to these phenotype.