HMA4 expression in tobacco reduces Cd accumulation due to the induction of the apoplastic barrier

Ectopic expression in tobacco (Nicotiana tabacum v. Xanthi) of the export protein AtHMA4 (responsible in Arabidopsis for the control of Zn/Cd root to shoot translocation) resulted in decreased Cd uptake/accumulation in roots and shoots. This study contributes to understanding the mechanisms underlying this Cd-dependent phenotype to help predict the consequences of transgene expression for potential phytoremediation/biofortification-based strategies. Microarray analysis was performed to identify metal homeostasis genes that were differentially expressed in roots of Cd-exposed AtHMA4-expressing tobacco relative to the wild type. It was established that down-regulation of genes known to mediate Cd uptake was not responsible for reduced Cd uptake/accumulation in AtHMA4 transformants. The transcript levels of NtIRT1 and NtZIP1 were higher in transgenic plants, indicating an induction of the Fe and Zn deficiency status due to AtHMA4 expression. Interestingly, upon exposure to Cd, genes involved in cell wall lignification (NtHCT, NtOMET, and NtPrx11a) were up-regulated in transformants. Microscopic analysis of roots demonstrated that expression of AtHMA4 caused an induction of cell wall lignification in the external cell layers that was accompanied by enhanced H2O2 accumulation. Further study showed that the concentration of other elements (B, Co, Cu, Ni, Mo, and Zn) was reduced in AtHMA4 transformants in the presence of Cd. In conclusion, due to ectopic expression of 35S::AtHMA4, the physical apoplastic barrier within the external cell layer developed, which is likely to be responsible for the reduction of Cd uptake/accumulation.