Genetic Isolation Of Cadmium Tolerance In Arabidopsis

Cadmium is a widespread nonessential toxic heavy metal,released into the biosphere mostly by modern industry.Phytochelatins(PCs)play an essential role in heavy-metal detoxification in plants and fungi.When exposed to heavy metal stress,PC biosynthesis is catalyzed by PC synthase(PCS)using glutathione as major substrates.The synthesized PCs then chelate heavy metals and the formed PC-metal complexes are translocated across the tonoplast and sequestered into vacuoles,thus decreasing the heavy-metal contents in the cytosol.AtPCS1 is the vital homologue that regulates PC synthesis and confers Arabidopsis constitutive tolerance to heavy metals.Loss of function in AtPCS1 (cad1-3)renders plants highly sensitive to Cd²⁺stress.In the present study,to obtain Cd tolerant mutants,a genetic screening was performed using EMS mutagenized cad1-3 plants.～10,000 cad1-3 mutant seeds were EMS mutagenized.Seeds from 6,000 M1 lines were harvested and partially pooled to make a library with 100,000 M2 seeds,among which about 40,000 M2 seedlings were determined by root bending assay.4 mutants were identified and assigned M38,M39,M46 and M51 respectively,which showed increased tolerance to Cd stress as determined by root bending assay.Preliminary genetic analyses showed that the 4 mutants were attributed to dominant mutations.To determine if gain of function in atpcs1-3 contributed to the phenotypes in M38,39,46 and 51,RT-PCR cloning and subsequent sequencing were performed.The results excluded the above possibility.Semiquantitative RT-PCR was also performed to determine expression level of AtPCS2. Interestingly,AtPCS2 expression was significantly increased in M38 and M39 compared to wild type Col-0 and cad1-3 mutant.Furthermore,the AtPCS2 expression is even greater in M38 than in M39,no significant change was observed in M46 compared to Col-0 and cad1-3.These results showed that heavy metal tolerance in M38,M39 might be partially attributed to modification of AtPCS2 expression.