Structure and function of metallothionein genes in Arabidopsis

Jianmin Zhou, Purdue University

Abstract

Metallothioneins (MTs) are cysteine-rich proteins required for heavy metal tolerance in animals and fungi. Five metallothionein genes, MT1a, MT1b, MT1c, MT2a, and MT2b, were cloned and characterized from Arabidopsis thaliana. At least four genes, MT1a, MT1c, MT2a and MT2b, were transcribed in Arabidopsis. These genes encode two groups of MTs, MT1 and MT2, that differ mainly in the presence or absence of a central domain. The Arabidopsis MTs are structurally homologous to animal and fungal MTs in their high cysteine content and arrangement of cysteine residues. The cDNAs for MT1a and MT2a were expressed in a MT-deficient (cup1$\sp\Delta$) mutant of yeast. Both MT1a and MT2a complemented the cup1$\sp\Delta$ mutation, providing a high level of resistance to CuSO$\sb4$ and moderate resistance to CdSO$\sb4$. While the MT-deficient yeast was not viable in the presence of either 300 $\mu$M CuSO$\sb4$ or 5 $\mu$M CdSO$\sb4$, cells expressing MT1 were able to grow in medium supplemented with 3 mM CuSO$\sb4$ or 10 $\mu$M CdSO$\sb4$, and those expressing MT2 grew in the presence of 3 mM CuSO$\sb4$ or 100 $\mu$M CdSO$\sb4$. When expressed in E. coli as glutathione S-transferase fusion proteins, both MT1a and MT2a bound copper, cadmium, and zinc. In plants, MT1 mRNA was more abundant in roots and dark-grown seedlings than in leaves. In contrast, MT2 mRNA accumulated more in leaves than in either roots or dark-grown seedlings. MT2 mRNA was strongly induced in seedlings by CuSO$\sb4$, but only slightly by CdSO$\sb4$ or ZnSO$\sb4$. However, MT1 mRNA was induced by CuSO$\sb4$ in excised leaves that were submerged in medium. The Arabidopsis MT genes were mapped to four loci on three chromosomes that are different from the location of CAD1, a gene responsible for cadmium tolerance in Arabidopsis. Furthermore, transgenic plants expressing either MT1a-antisense or MT2a-antisense RNAs showed increased sensitivity of root growth to copper but not to cadmium. These results indicate that Arabidopsis MT genes are specifically involved in tolerance to copper. Plants also synthesize metal-binding phytochelatins (poly ($\gamma$-glutamylcysteine) glycine) when exposed to heavy metals. The results presented here argue against the hypothesis that phytochelatins are the sole molecules involved in heavy metal tolerance in plants. We conclude that Arabidopsis MT1 and MT2 are functional homologs of yeast MT.

Degree

Ph.D.

Advisors

Goldsbrough, Purdue University.

Subject Area

Molecular biology|Genetics

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