Identiﬁcation and characterization of several new members of the ZIP
family of metal ion transporters in Medicago truncatula
n, Danielle R. Ellis and Michael A. Grusak*
Department of Pediatrics, USDA/ARS Children’s Nutrition Research Center, Baylor College of Medicine,
1100 Bates Street, Houston, TX 77030, USA (*author for correspondence; e-mail firstname.lastname@example.org)
Received 31 July 2003; accepted in revised form 15 March 2004
Key words: gene expression, Medicago truncatula, metals, plasma membrane, transporters, ZIP genes
To broaden our understanding of micronutrient metal transport in plants, we have identiﬁed cDNAs for six
new metal transporters in the model legume Medicago truncatula. All of the predicted proteins have high
similarity to the ZIP protein family, and have been designated MtZIP1, MtZIP3, MtZIP4, MtZIP5,
MtZIP6, and MtZIP7. The six predicted proteins ranged from 350 to 372 amino acids in length; sequence
analysis revealed that all proteins contained eight transmembrane domains and the highly conserved ZIP
signature motif. Most of the proteins also exhibited a histidine-rich region in the variable sequence between
transmembrane domains III and IV. When MtZIPs were transformed into appropriate metal-uptake
defective yeast mutants and grown on metal-limited media, MtZIP1, MtZIP5, and MtZIP6 proteins re-
stored yeast growth on Zn-limited media, MtZIP4 and MtZIP7 proteins restored yeast growth on Mn-
limited media, and MtZIP3, MtZIP5, and MtZIP6 proteins restored yeast growth on Fe-limited media.
Therefore, we conclude that these proteins function as metal transporters in Medicago truncatula. The
expression pattern for each gene was studied by semi-quantitative RT-PCR in roots and leaves from plants
grown under various metal supplies. MtZIP1 transcripts were only detected in Zn-deﬁcient roots and
leaves. MtZIP3 and MtZIP4 expression was down regulated in leaves from Mn- and Fe-deﬁcient plants
and appeared to be upregulated under Zn-deﬁcient conditions in both roots and leaves. MtZIP5 was
upregulated in leaves under Zn and Mn deﬁciency. The expression of MtZIP6 and MtZIP7 was unaﬀected
by the metal supply, at least in root and leaf tissues. Characterizing these proteins in a single organism will
allow us to understand the interplay between various ZIP genes, and the role they play in the regulation/
execution of plant metal homeostasis.
Plants require metals such as Fe, Zn, and Mn for
essential functions ranging from respiration to
photosynthesis, as they are requisite components or
cofactors of many key enzymes in these physio-
logical processes (Marschner, 1985). Consistent
with the fundamental role of these elements, deﬁ-
ciency causes severe disorders in plants. In general,
metal deﬁciencies cause an altered expression and
or function of proteins at the metabolic level that
leads to diﬀerent physiological symptoms such as
reduced growth, leaf necrosis and chlorosis
(Kriedmann et al., 1985; Terry and Abadia, 1986).
On the other hand, excessive amounts of these
metals can cause serious damage to the plant, due to
uncontrolled binding of the metals to proteins (Van
Assche and Clijsters, 1986a, b) or due to their redox
activity that can lead to the generation of oxygen
radicals (Pahlsson, 1989; Price and Hendry, 1991).
Therefore, plants must carefully regulate both me-
tal acquisition from the soil and cellular partition-
ing within the plant in order to prevent excess
accumulation while obtaining adequate intake.
Plant Molecular Biology 54: 583–596, 2004.
Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands.