Ancient classes of metallothioneins in Arabidopsis thaliana
Zimeri, Anne Marie Swartz
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Metallothioneins (MTs) are small metal-binding proteins, typically less than 85 amino acids, which appear to be ubiquitous in eukaryotes and cyanobacteria. The Arabidopsis genome contains 9 putative metallothionein (MT) sequences with classical cysteine rich domains separated by spacer sequences. Phylogenic analysis of these and other plant MTs from numerous species identified four ancient classes (MT1-4) of MT sequences that predate the monocot-dicot plant group divergence 200 million years ago. The selective pressure that preserved these ancient classes of MTs in the plant genome is likely due to distinct functions, such as differential metal binding properties that provide protection from toxic metals and elevated levels of nutrient metals. Contrary to the current tenet that MTs bind metals in accordance with the thiolate series, and consistent with the hypothesis that MTs have distinct functions, differential in vivo stabilization by metal ions was found among representatives of the four ancient MT classes. For example, based on protein stability, MT1 is best stabilized by cadmium followed closely by copper and zinc. In contrast, MT2 is best stabilized by cadmium followed by arsenic, MT3 is best stabilized by zinc followed by cadmium, and MT4 is best stabilized by cadmium followed by copper. RNA interference (RNAi) was used to disrupt translation of the entire Arabidopsis MT1 class to address its function in plants. Based on phylogenetic and protein stability data, it was hypothesized that suppression of all three MT1 class members would render Arabidopsis plants hypersensitive to cadmium. Plants with knocked down MT1 expression were found to be severely inhibited by cadmium as compared to wildtype. In addition, these lines accumulated less cadmium per gram of tissue than wildtype as determined by inductively coupled plasma mass spectrometry. Based on the protein stabilization data for MT3, we wanted to determine if overexpression of MT3 would generate plants with increased zinc composition. Surprisingly, severe zinc sensitive phenotypes were observed in plants when zinc sulfate was added to germination media. However, plants germinated on normal growth media, then transferred to media supplemented with zinc, exhibited no growth or germination phenotypes, nor did they accumulate more zinc than wildtype controls. This suggests the mechanisms of sensitivity may be due to a maternal effect, and that mitochondria or chloroplasts are responsible for the observed phenotype. In addition to phylogenetic, metal stabilization, and in vivo functional experiments, the characterization of Arabidopsis MTs in this body of work has identified candidate molecules toward both phytoremediation and nutrient enhancement ends. The field of phytoremediation uses both natural and engineered plants to detoxify the soil by both altering the state of the toxin to a less harmful form and/or by hyperaccumulating toxins that can be physically removed by harvesting the plant. Molecules that bind specific metals, like MTs, have great potential for use in hyperaccumulating heavy metals in plant tissues. Genetically engineered plants that accumulate metals promise to improve agriculture as well. Crop plants that can accumulate nutrient metals, such as zinc, can benefit human health. In addition, plants engineered to better tolerate metals present in the soils can potentially expand geographic areas where plants can be grown.