title: Barley metallothioneins: MT3 and MT4 are localized in the grain aleurone layer and show differential zinc binding.
abstract: Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins believed to play a role in cytosolic zinc (Zn) and copper (Cu) homeostasis. However, evidence for the functional properties of MTs has been hampered by methodological problems in the isolation and characterization of the proteins. Here, we document that barley (Hordeum vulgare) MT3 and MT4 proteins exist in planta and that they differ in tissue localization as well as in metal coordination chemistry. Combined transcriptional and histological analyses showed temporal and spatial correlations between transcript levels and protein abundance during grain development. MT3 was present in tissues of both maternal and filial origin throughout grain filling. In contrast, MT4 was confined to the embryo and aleurone layer, where it appeared during tissue specialization and remained until maturity. Using state-of-the-art speciation analysis by size-exclusion chromatography inductively coupled plasma mass spectrometry and electrospray ionization time-of-flight mass spectrometry on recombinant MT3 and MT4, their specificity and capacity for metal ion binding were quantified, showing a strong preferential Zn binding relative to Cu and cadmium (Cd) in MT4, which was not the case for MT3. When complementary DNAs from barley MTs were expressed in Cu- or Cd-sensitive yeast mutants, MT3 provided a much stronger complementation than did MT4. We conclude that MT3 may play a housekeeping role in metal homeostasis, while MT4 may function in Zn storage in developing and mature grains. The localization of MT4 and its discrimination against Cd make it an ideal candidate for future biofortification strategies directed toward increasing food and feed Zn concentrations.
authors.affiliation: Plant and Soil Science Section, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, DK-1871 Frederiksberg, Denmark.
authors.forename: Josefine Nymark
authors.initials: JN
authors.lastname: Hegelund
authors.forename: Michaela
authors.initials: M
authors.lastname: Schiller
authors.forename: Thomas
authors.initials: T
authors.lastname: Kichey
authors.forename: Thomas Hesselhøj
authors.initials: TH
authors.lastname: Hansen
authors.forename: Pai
authors.initials: P
authors.lastname: Pedas
authors.forename: Søren
authors.initials: S
authors.lastname: Husted
authors.forename: Jan Kofod
authors.initials: JK
authors.lastname: Schjoerring
mesh_terms: D000222:Adaptation, Physiological / Q000187:drug effects; D000595:Amino Acid Sequence; D002104:Cadmium / Q000633:toxicity; D002850:Chromatography, Gel; D003300:Copper / Q000633:toxicity; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D001467:Hordeum / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D013058:Mass Spectrometry; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism; D012639:Seeds / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D016415:Sequence Alignment; D015032:Zinc / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D002104:Cadmium; D003300:Copper; D008668:Metallothionein; D015032:Zinc