Arabidopsis metallothioneins 2a and 3 enhance resistance to cadmium when expressed in Vicia faba guard cells

The Arabidopsis metallothionein genes AtMT1 and AtMT2 confer Cd(II) resistance to Cd(II)-sensitive yeast, but it has not been directly shown whether they or other metallothioneins provide the same protection to plants. We tested whether AtMT2a and AtMT3 can confer Cd(II) resistance to plant cells by introducing GFP- or RFP-fused forms into guard cells of Vicia faba by biolistic bombardment. AtMT2a and AtMT3 protected guard cell chloroplasts from degradation upon exposure to Cd(II), an effect that was confirmed using an FDA assay to test the viability of the exposed guard cells. AtMT2a- and AtMT3-GFP were localized in the cytoplasm both before and after treatment of V. faba guard cells or Arabidopsis protoplasts with Cd(II), and the levels of reactive oxygen species were lower in transformed guard cells than in non-transformed cells after Cd(II)-treatment. These results suggest that the Cd(II)-detoxification mechanism of AtMT2a and AtMT3 may not include sequestration into vacuoles or other organelles, but does involve reduction of the level of reactive oxygen species in Cd(II)-treated cells. Increased expression of AtMT2a and AtMT3 was observed in Arabidopsis seedlings exposed to Cd(II). Together, these data support a role for the metallothioneins AtMT2a and AtMT3 in Cd(II) resistance in intact plant cells.