Download Expression of the Vacuolar Iron Transporter

Expression of the Vacuolar Iron Transporter-Like Genes and their Localization
in Saccharomyces and Arabidopsis
Roman Timofeev*, Zahra Ghalamkari*, Thomas J. Buckhout**
Applied Botany, Institute of Biology, Humboldt University, Invalidenstraße 42, 10115 Berlin, Germany
*These authors have contributed equally to this report.
**Corresponding Author: [email protected]
Iron deficiency is a nutritional problem in plants and reduces crop productivity and quality. We have
investigated the function of a small family of five membrane proteins that have amino acid sequence
homology to AtVIT1 and consequently also to yeast CCC1p (1-3). These Vacuolar-Iron-Transporterlike (VTL) genes are found both in mono‐ and dicotyledon plants, as well as in the green alga
Chamydomonas and the moss Physcomitrella. With the goal of further characterizing the function of
these proteins and improving the Fe storage properties of plants, we have investigated the localization
of the VTL proteins in Saccharomyces and the effects of their over-expression in Arabidopsis.
AtVTL1 and AtVTL2 have been localized on the vacuolar membrane of onion epidermis cells that were
transiently transformed with 35S:GFP::VTL1 or 35S:GFP2::VTL2 (3). However, none of the chimeric
constructs showed complementation of the yeast Δccc1 mutant. When a flexible linker was inserted
between the VTL and GFP genes (pPGK:VTL::FL3::GFP) all five genes complemented the yeast
Δccc1 mutant. The complementation activity was, however, significantly weaker than with the VTL
genes alone (3). We demonstrate here for the first time that VTL3 (pPGK:VTL3::FL3::GFP) and VTL4
(pPGK:VTL4::FL3::GFP) were clearly localized to the vacuolar membrane in yeast. In contrast
however, the origin of the fluorescent signal observed in cells transformed with the VTL5 gene
(pPGK:VTL5::FL3::GFP) was ambiguous. Although some cells showed labeling of the vacuolar
membrane, labeling of the endoplasmic reticulum, trans-Golgi network and plasma membrane were
most frequently observed. VTL5 was expressed throughout the plant in the vegetative shoot apex, in
response to biotic stress and in the root endodermis (source: Arabidopsis eFP Browser). We have
investigated the expression of VTL5 using promotor-GUS assays in stably transformed Arabidopsis
seedlings (pVTL5:GUS). Under the conditions of excess iron supply (120 μM), VTL5 was expressed
predominately in the root.
The VTL genes have been over-expressed in Arabidopsis and the iron content in these transgenic
plants has been investigated. The iron content of seeds of VTL over‐ expressing plants was measured
and simultaneously the expression of these genes analyzed. The results showed a correlation
between overexpression of VTL2 and VTL5 with the Fe content in seeds. The Fe content in the seeds
of the VTL2 overexpressing lines was ca. 6.4 nmole/mg dry mass and in the VTL5 overexpressor 6.3
nmole/mg, whereas in the wild‐ type plant the Fe concentration was ca. 3.3 nmole/mg dry mass. The
embryos from the wild‐ type and overexpressing plants were dissected from seeds and stained by the
Perls’ method with DAB intensification. These Embryos showed the localization of Fe in the vascular
system in both overexpressing and wild‐ type plants. In VTL5 overexpressing plants, staining was
more intense throughout the embryo and particularly at the tip of radicle compare with wild‐ type plant.
The regulation of expression of VTL genes by iron and the localization of their expression indicated an
important role of the VTLs in iron homeostasis in the plant Arabidopsis.
Keywords: Biofortification, CCC1, Iron, Transport, Vacuole
REFERENCES
1. Buckhout TJ et al. BMC Genomics. 2009, 6(10):147
2. Gollhofer J et al. Plant Physiol Biochem. 2011, 49(5): 557
3. Gollhofer J et al. PLoS One. 2014, 9(10): e110468
ACKNOWLEDGEMENTS
The support of the DFG and Jameel Foundation is gratefully acknowledged.