Functional characterization of seed-expressed metallothioneins in Arabidopsis thaliana

Napassorn Punyasuk, Purdue University

Abstract

Metallothioneins (MTs) are low-molecular-weight (4-8 kDa), cysteine-rich proteins that can bind metals via their thiol groups. Of the seven MT genes in Arabidopsis, five are expressed predominantly in vegetative tissues whereas the two Type 4 MTs are expressed only in seeds. The functions of MTs in seeds are unknown. I hypothesize that seed-expressed MTs participate in accumulation of metal ions (Cu and Zn) and their utilization during seed germination and early seedling development. T-DNA insertion mutants and MT4 overexpressors were used to study the functions of seed-expressed MTs. Mutants that are deficient in seed-expressed MTs, including mt2a, mt4a, mt4b single mutants, the mt4a/mt4b double mutant and the mt2a/mt4a/mt4b triple mutant plants grow normally under standard conditions and show no significant change in the concentrations of metals in seeds. To test their response to more extreme conditions of metal deficiency or excess, the mt2a/mt4a/mt4b triple mutant was also grown under deficient or excess of Zn. However, there were no significant differences in seed Zn content between wild-type and the mt2a/mt4a/mt4b triple mutant under these metal treatments. Similar results were obtained when the triple mutant was grown under deficient or excess of Cu. These results indicate that these MTs that are expressed in seeds do not play a major role in the accumulation of Zn and Cu in seeds. When grown under Cu-deficient conditions in a hydroponic system, mutants that lack seed-expressed MTs grow slower and have smaller rosettes than wild-type indicating that these mutants are sensitive to Cu deficiency. Analysis of some genes that are regulated by Cu status, including CSD1 and COPT2, also indicates that mutants had a normal response to Cu deficiency. A similar reduction in growth was observed when these mutants were grown on agar medium with a Cu chelator, ammonium tetrathiomolybdate (TM). Complementation lines, including mt4a+MT4a, mt4b+MT4b, mt4a/mt4b+MT4a and mt4a/mt4b+MT4b, showed improved growth under Cu-deficient conditions indicating that the sensitivity of mt4a and mt4b mutants to Cu deficiency was caused by the lack of MT4s. When seeds of the mt2a/mt4a/mt4b triple mutant with low Zn content were grown on medium that contained no Zn and no sucrose, they germinated but did not grow further and seedling development was arrested. Growth of these seedlings was restored if they were transferred to medium with sucrose. This result suggests that under Zn deficient conditions, seed-expressed MTs are important for establishing photosynthetic capacity in young seedlings. Constitutive expression of Type 4 MT genes driven by the 35S promoter results in increased accumulation of Cu and Zn in both seeds and shoots, and hypersensitivity to Zn. MT4 overexpressors also showed hypersensitivity to Cu deficient conditions. These results indicated that ectopic expression of Type 4 MT has a negative impact on Cu and Zn homeostasis. In summary, characterization of plants with altered expression of seed-expressed MTs provides evidence that these proteins play important roles in metal homeostasis during germination and early seedling development.

Degree

Ph.D.

Advisors

Goldsbrough, Purdue University.

Subject Area

Botany|Genetics|Plant sciences

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