Analysis of Cd tolerance/accumulation and identification of the related genes in a metal hyper-tolerator fern, Athyrium yokoscense
The mechanism of Cd tolerance/accumulation of a metal hyper-tolerator fern, Athyrium yokoscense, was analyzed at physiological and molecular views using the callus. Another callus induced from tobacco plants, which shows comparatively high Cd tolerance/accumulation ability among angiosperms, was used as a control. The fern callus grew vigorously even under the considerably high concentration of Cd (100M) in a liquid culture system, although the tobacco callus reduced the growth at the condition for 20% of the control condition. More than 90% of the total cellular Cd accumulation was distributed in the cell wall fraction in the fern, while only about 40% of that was in the fraction in tobacco plants. Further observations using protoplast verified the result; the fern protoplasts contained only one-fifth absolute amount of Cd than the tobacco protoplasts. Furthermore, the six essential-metal concentrations (Ca, Cu, Fe, Mg, Mn and Zn) were determined in those calli grown under the Cd condition using ICP. The tissue concentration of Cu, Fe and Zn were almost constant, whereas that of Ca, Mg and Mn was slightly but significantly decreased in the fern callus. On the contrary, that of Fe was drastically decreased (more than 50%) but the others were almost constant in the tobacco callus. Thus, the fern callus could grow normally even under the Cd-exposure condition, although the tobacco callus was in a state of Fe-deficiency. These observations indicate two possibilities that essential-metal transporters, especially Fe-transporters, of the fern have high selectivity for the targeted metals even under the Cd-exposure condition and/or Cd once imported into cells excreted by a hypothetical exporter. Thus, either or both of these may induce the fact that low Cd concentration in the inner cell membrane of the fern, and relate to at least a part of the Cd tolerance of the fern. Subsequently, four Fe-transporters (AyIRT1, AyNramp1, AyYSL1 and AyYSL2), which has already been confirmed to transport Fe in some plant species and may relate to the Cd transport, were isolated from the fern. Because two of those genes, AyIRT1 and AyNramp1, contained full of the ORF, those were characterized for their putative proteinaceous structures and overexpressed in a yeast mutant strain, which is deficit in a part of Fe-acquisition ability. Although each of the putative structures consist of lesser numbers of trans-membrane domains than known structures of their Arabidopsis homologues, it conserves major common amino acid sequences, which may relate to the Fe transport. Both types of the yeast overexpressors showed higher Cd tolerance with less amount of cellular Cd concentration.
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