75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

127 AtCKT1, a Novel Transporter for Purines and Cytokinins in Arabidopsis thaliana
Benjamin Schumacher 2 , Veronica Maurino 1 , Esther Grube 1 , Ulf Flugge 1 , Marcelo Desimone 2
1
Botanical Institute, University of Cologne, Germany, 2 ZMBP, Plant Physiology, University of Tuebingen,
Germany
Cytokinins (CK) allocation plays a key role in plant development. Both xylem and phloem serve for long distance
transport of CK, but the mechanisms involved in their loading at the site of synthesis and unloading at the site of
utilization remains unclear. A family of purine permeases (PUPs) and a family of nucleoside transporters (ENTs) are
able to transport CK free bases and nucleosides respectively, suggesting that plants utilizes transporters with broad
specificities for CK movement through cell membranes. In this work, a novel transporter of Arabidopsis was identified
(AtCKT1). It belongs to a protein family with a member recently characterized in Aspergillus (AzgA1) and shows high
homology to several proteins encoded in plant genomes. When expressed in a yeast mutant defective in adenine uptake
(fcy2), AtCKT1 restored the capacity for adenine transport. Competition experiments indicate that CK are also potential
substrates. Adenine uptake into yeast cells by AtCKT1 is coupled to proton transport as suggested by pH dependency
and proton gradient inhibitors. Transient expression of GFP-AtCKT1 protein fusions in Arabidospis cells indicates
localization of AtCKT1 in the plasma membrane. Transgenic plants carrying the reporter gene GUS under control of the
AtCKT1 promoter and RT-PCR analysis revealed that AtCKT1 is mainly expressed in roots at relative low levels. T-DNA
insertion lines producing knock out of AtCKT1 are resistance to toxic purine analogs and present lower sensitivity to CK
application. In contrast, plants overexpressing AtCKT1 showed enhanced sensitivity to toxic purine analogs and CK,
and increased capacity to adenine uptake from the environment. These data suggest that AtCKT1 is involved in purine
and CK metabolism in Arabidopsis roots.
128 Genetic Analysis of SCY1 and SCY2 Function in Arabidopsis
Courtney Skalitzky, Jessica Harwood, Gregory Heck, Donna Fernandez
University of Wiscosin-Madison
SCY1 and SCY2 are plastid localized homologs of the bacterial SecY protein. The SecY translocon in the plasma
membrane of eubacteria facilitates both the secretion of peptides out of the cell and the insertion of proteins into the lipid
bilayer. In addition to one subunit of SecY, the translocon contains one subunit of SecE and SecG. Plastids contain SecY
and SecE homologs, but not SecG. Two SecY homologs have been found in Arabidopsis, which we have designated as
SCY1 (At2g18710) and SCY2 (At2g31530). Both SCY1 and SCY2 have ten predicted transmembrane domains and their
amino acid sequences are only 28% identical and 44% similar. Previous studies showed SCY1 is targeted to the thylakoid
membranes in the chloroplast. SCY2 sequence includes a putative transit peptide and import studies have shown that it
is also targeted to the plastids. RT-PCR analysis revealed that SCY1 and SCY2 are expressed in both shoots and roots
and both genes are constitutively expressed in the light and the dark. Despite these similarities, genetic analyses indicate
that they play different roles in plant development. Mutations in SCY1 cause a seedling lethal phenotype, while mutations
in SCY2 result in embryo lethality. To investigate whether expression differences account for functional differences, we
performed promoter swap experiments. Based on our current results, we conclude that differences in function are more
likely to form the basis of the different mutant phenotypes than expression differences.
One possibility that we are considering is that SCY1 and SCY2 function in different membranes within plastids.
We are using SCY2-GFP protein fusions to determine the subcellular localization of SCY2. Supported by UW-Madison
Graduate School.