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.

Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis

“…The expression and localization of MTs vary between tissues and organs. In both Arabidopsis (Arabidopsis thaliana) and barley (Hordeum vulgare), MT4 isoforms contribute to the storage and release of Zn 2+ during embryogenesis (Hegelund et al, 2012;Ren et al, 2012 Fig. S4).…”

Got to hide your Zn away: Molecular control of Zn accumulation and biotechnological applications

“…Over-expression of AtMT4a improved Zn tolerance in A. thaliana mature plants as well [23]. The barley single type 4 metallothionein protein has much higher affinity for Zn binding than for Cd, and is specifically expressed in the embryo and aleurone layer [24]. Over-expressing OsMT1a in rice resulted in increased accumulation of Zn in both leaves and grains [25].…”

“…The metallothionein 4 protein from barley has large Zn-binding capacity and may function in Zn storage in developing and mature grains [24]. Metallothionein 4 is confined to the barley embryo and aleurone layer [24] as well as into Arabidopsis embryos and developing seeds, making type 4 metallothioneins interesting candidates for future Zn biofortification work.…”

Zinc (Zn) Uptake

“…9.4.1.6.3). It has been reported that MT4 confined to the embryo and aleurone layer in barley grains is involved in preferential Zn binding and functions in Zn storage in developing and mature grains (Hegelund et al 2012). …”