A tobacco plasma membrane calmodulin-binding transporter confers Ni²⁺ tolerance and Pb²⁺ hypersensitivity in transgenic plants

All organisms require a minimal amount of metal ions to sustain their metabolism, growth and development. At the same time, their intrinsic metal-uptake systems render them vulnerable to toxic levels of metals in the biosphere. Using radiolabeled recombinant calmodulin as a probe to screen a tobacco cDNA library, we have discovered a protein designated NtCBP4 (Nicotiana tabacum calmodulin-binding protein) that can modulate plant tolerance to heavy metals. Structurally, NtCBP4 is similar to vertebrate and invertebrate K⁺ and to nonselective cation channels, as well as to recently reported proteins from barley and Arabidopsis. Here we report on the subcellular localization of NtCBP4 and the phenotype of transgenic plants overexpressing this protein. The localization of NtCBP4 in the plasma membrane was manifested by fractionating tobacco membranes on sucrose gradients or by aqueous two-phase partitioning, and subsequently using immunodetection. Several independent transgenic lines expressing NtCBP4 had higher than normal levels of NtCBP4. These transgenic lines were indistinguishable from wild type under normal growth conditions. However, they exhibited improved tolerance to Ni²⁺ and hypersensitivity to Pb²⁺, which are associated with reduced Ni²⁺ accumulation and enhanced Pb²⁺ accumulation, respectively. To our knowledge this is the first report of a plant protein that modulates plant tolerance or accumulation of Pb²⁺. We propose that NtCBP4 is involved in metal uptake across the plant plasma membrane. This gene may prove useful for implementing selective ion tolerance in crops and improving phytoremediation strategies.