UC San Diego

Heavy metal pollution is a global environmental concern. Plants have been discovered at contaminated sites, however, the molecular mechanisms that these plants employ to deal with heavy metal stress is not well characterized. To gain a further insight into the genes and molecular pathways plants utilize to deal with cadmium stress, a post- germination screen was developed in the plant model system Arabidopsis to isolate mutants tolerant to high levels of cadmium. Several cadmium tolerant mutants were isolated from screening EMS mutagenized and activation tagged populations. Cd11, an EMS mutagenized mutant, displayed a significantly increased tolerance to cadmium in both shoot and root tissues in comparison to wild type and over- accumulated cadmium in both shoot and root tissues in comparison to wild type. Several activation tagged mutants were also shown to exhibit increased tolerance to cadmium in both shoot and root tissues. One of the primary mechanisms that plants employ for dealing with heavy metal stress is the production of phytochelatins. Phytochelatins are produced post-translationally by the enzyme phytochelatin synthase (PCS). Arabidopsis has two functional AtPCS genes. We have isolated a loss-of- function allele in the AtPCS2 gene, atpcs2-1, and generated a double loss-of-function AtPCS mutant, cad1-3 atpcs2-1. atpcs2-1 did not show an altered response to cadmium in comparison to wild type in either shoot or root tissues, cadmium accumulation, or levels of phytochelatins. cad1-3 atpcs2-1 shared a similar response to cadmium as the single mutant cad1-3 in shoot and root growth, cadmium accumulation, and the lack of detectable phytochelatins. In addition, we have determined that phytochelatins were able to undergo long distance transport in a shoot to root direction. Fluorescence HPLC coupled to mass spectrometry analyses and detected the presence of phytochelatins in root tissues of transgenic plants expressing the wheat PCS (TaPCS1 ) gene under the control of a shoot specific promoter (CAB2 ) in the PC- deficient mutant cad1-3. Analyses demonstrated that CAB2:: TaPCS1/cad1-3 lines complement the cadmium and arsenic metal sensitivity of cad1-3 shoots and over-accumulated cadmium in root tissues. Grafting experiments between wild type shoots and cad1-3 atpcs2-1 roots also showed shoot to root PC transport