Engineering copper hyperaccumulation in plants by expressing a prokaryotic copC gene

In this work, engineering Cu-hyperaccumulation in plants was approached. First, the copC gene from Pseudomonas sp. Az13, encoding a periplasmic Cu-binding protein, was expressed in Arabidopsis thaliana driven by the CaMV35S promoter (transgenic lines 35S-copC). 35S-copC lines showed up to 5-fold increased Cu accumulation in roots (up to 2000 μg Cu. g(-1)) and shoots (up to 400 μg Cu. g(-1)), compared to untransformed plants, over the limits established for Cu-hyperaccumulators. 35S lines showed enhanced Cu sensitivity. Second, copC was engineered under the control of the cab1 (chlorophyll a/b binding protein 1) promoter, in order to drive copC expression to the shoots (transgenic lines cab1-copC). cab1-copC lines showed increased Cu translocation factors (twice that of wild-type plants) and also displayed enhanced Cu sensitivity. Finally, subcellular targeting the CopC protein to plant vacuoles was addressed by expressing a modified copC gene containing specific vacuole sorting determinants (transgenic lines 35S-copC-V). Unexpectedly, increased Cu-accumulation was not achieved-neither in roots nor in shoots-when compared to 35S-copC lines. Conversely, 35S-copC-V lines did display greatly enhanced Cu-hypersensitivity. Our results demonstrate the feasibility of obtaining Cu-hyperaccumulators by engineering a prokaryotic Cu-binding protein, but they highlight the difficulty of altering the exquisite Cu homeostasis in plants.