Functional Characterization of Metallothioneins in Arabidopsis and Barley

Metalothioneins (MTs) are metal binding proteins that can bind metals such as Cu, Zn, and Cd but the functions of MTs in plants are largely unknown. To understand the function of MTs in Arabidopsis, T-DNA insertion mutants lacking 4 MT genes including MT1a, MT2a, MT2b and MT3 were developed. The quadruple mutant (mt1a/mt2a/mt2b/mt3) showed no visible phenotype under standard growth conditions. However, it accumulated higher Cu in leaves compared to wild type but lower Cu in seeds. Further analysis showed that triple mutants lacking both MT1a and MT2b, which are highly expressed in vascular tissues, had similar dramatic changes in Cu accumulation in leaves and seeds, suggesting that MTs expressed in vasculature are critical for metal distribution in leaf and seed tissues. In wild type leaves, the concentration of Cu declined as the leaves began to senescence whereas Cu increased in the quadruple mutant. Experiments with stable isotopes of Cu showed no difference in the rate of Cu uptake between wild type and quadruple mutant plants. Therefore, high accumulation of Cu in the quadruple mutant leaves is likely due to reduced remobilization of Cu out of leaves. Increased Cu accumulation in leaves of the quadruple mutant was also accompanied by altered RNA expression of several genes involved in Cu homeostasis including FSD1, CSD1, COPT2, YSL2, and HMA5. Although MTs are important for mobilization of Cu out of senescing leaves, this does not make a major contribution to the Cu that accumulates in seeds. Experiments with stable isotopes of Cu showed that most Cu in seeds was uploaded directly from the nutrient solution. Inflorescence grafting experiments demonstrated that MTs are involved in the short range Cu translocation from maternal tissues into developing seeds but not in the long distance transport from leaves and roots. Thus, the high accumulation of Cu in leaves and low accumulation in seeds in MT-deficient plants are independent processes. The Arabidopsis quadruple mutant was sensitive to Cu deficiency with reduced root growth and interveinal chlorosis in mature rosette leaves. The sensitivity to Cu deficiency was even more severe when plants were grown under high light conditions. Under conditions of reduced Cu availability, the quadruple mutant still accumulated more Cu in shoots than WT plants. The leaf phenotype did not appear in any of single gene MT mutants, indicating that MTs have redundant functions. The triple mutants lacking both MT2a and MT2b showed leaf chlorosis similar to the quadruple mutant, suggesting these MT genes are important for this phenotype. Analysis of genes that are involved in Cu homeostasis including FRO5, YSL2 and YSL3 showed that the quadruple mutant plants were less sensitive to Cu deficiency in roots and had low availability of Cu for remobilization in shoots compared to WT plants. Withdrawing Cu supply during reproductive stage also caused severe effects on flower and seed development in the quadruple mutant. In barley, I identified 9 barley MT genes from EST databases. Study of RNA expression demonstrated that MT gene expression is tissue specific. When barley plants were grown under various concentration of Cu and Zn, MT2a, MT2b and MT3 were up-regulated in roots under high Cu and MT1c was up-regulated under high Zn, indicating the connection between MTs and metal homeostasis. Virus induced gene silencing (VIGS) was used to study the function of MT1a in barley; however, the virus could not silence the expression of this gene. Further investigation on the stability of the MT RNA fragment in the VIGS virus will be necessary. Taken together, MTs play critical roles in Cu homeostasis, especially Cu accumulation in leaves and seeds. These data will provide a better understanding of how plants maintain metal homeostasis in their organs