Molecular genetics of Arabidopsis metallothioneins: Function and gene regulation

Metha Meetam, Purdue University

Abstract

Metallothioneins (MTs) are small cysteine-rich metal-binding proteins found in most eukaryotes and a few bacteria. Four conserved classes of MTs have been identified in angiosperms, but their functions remain enigmatic. In Arabidopsis thaliana, mRNAs of MT2a, MT4a and MT4b are abundant in seeds. RNA blot and RT-PCR showed that MT4a and MT4b mRNAs accumulated during late embryogenesis and disappeared rapidly following seed imbibition; MT2a mRNA was detected in all developmental stages. In mature embryos, GUS expression directed by the MT2a promoter was localized in the cotyledons whereas the MT4a and MT4b promoters were active in all tissues. When expressed in metal-sensitive yeast mutants, MT4a and MT4b were equally able to provide copper tolerance, but MT4a was more efficient than MT4b in conferring zinc tolerance. Functions of these MTs were investigated using Arabidopsis mutants that lacked MT2a, MT4a, MT4b, or both MT4a and MT4b. Seed development and germination were not affected by the mutations. However, seeds of all mutant genotypes contained reduced amounts of copper compared to the wild-type level. A small but significant reduction in zinc content was observed in seeds from the mt4a and mt4a/mt4b mutants. These results suggest that MTs play an important role in accumulation of copper and zinc during seed development. Expression of the MT2a gene in vegetative tissues is inducible by copper and oxidative stress. Analysis of the MT2a promoter identified sequences from -193 to -130 upstream of the transcriptional initiation site that were essential for basal expression and induction of the gene in response to 50 μM CuSO4 or 10 mM H2O 2. When placed in front of a minimal promoter, the -201 to -126 MT2a promoter sequence was also sufficient to confer high-level responsiveness to Cu and H2O2. To identify trans-acting regulatory factors that mediate copper induction of the MT2a gene, a small population of EMS-mutagenized Arabidopsis (expressing MT2a::GUS) was screened for mutants with altered MT2a expression and/or tolerance to copper. Six mutants were identified. Preliminary characterization showed that the lrb25 mutant contained a recessive mutation leading to constitutive MT2a expression in the root and increased tolerance of root growth to copper toxicity.

Degree

Ph.D.

Advisors

Goldsbrough, Purdue University.

Subject Area

Molecular biology|Botany

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