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

255 Cell type-specific expression and boron-dependent endocytosis of BOR1, a boron
transporter
Junpei Takano 1 , Kyoko Miwa 1 , Toru Fujiwara 1, 2
1
Biotechnology Research Center, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, 2 SORST, JST
Boron (B) is essential for plants and has a structural role in cell wall. Besides its essentiality, B is toxic when present
in excess. Arabidopsis thaliana BOR1 is a B exporter for xylem loading and is essential for efficient B translocation
from roots to shoots under B limitation (Takano et al. 2002). Using transgenic plants expressing BOR1-GFP fusion
protein under control of the cauliflower mosaic virus 35S RNA promoter, we have demonstrated that posttranslational
mechanisms play a major role in regulation of BOR1 accumulation. In the root tip cells of the 35S:BOR1-GFP plants,
BOR1-GFP was localized to the plasma membrane under B limitation and was transferred via the endosomes to the
vacuole for degradation upon exposure to high levels of B (Takano et al. 2005).
Here we report cell-type specific expression and boron-dependent endocytosis of BOR1-GFP expressed under control
of the BOR1 promoter. BOR1-GFP was localized to steler cells in mature portion of roots and also to various cells in
root elongation zone under B limitation. These results suggest that BOR1 functions in roots are zone specific. BOR1
functions for B transport into xylem in mature portion for root-to-shoot B translocation while it is for radial B transport
towards inner portions in root elongation zone. We have recently shown that T-DNA insertion mutants of a boric acid
channel NIP5;1 and a double insertion mutant of B exporters BOR1/BOR2 were defective in root cell elongation under B
limitation (Takano et al. 2006, Miwa et al. unpublished results). Taken together, our results suggest that radial transport
of B by these transporters are important for supplying B to elongating cell walls under B limitation.
Moreover, closer observation of epidermal cells in elongation zone revealed that the BOR1-GFP was disappeared from
plasma membrane upon exposure to high levels of B. We propose that the B-dependent endocytosis of BOR1 provides
a fast and efficient way to control B transport necessary under B limitation but detrimental under high B supply.
Takano et al. (2002) Arabidopsis boron transporter for xylem loading. Nature 420: 337-340
Takano et al. (2005) Endocytosis and degradation of BOR1, a boron transporter of Arabidopsis thaliana, regulated by boron availability. PNAS
102: 12276-12281
Takeno et al. (2006) The Arabidopsis major intrinsic protein NIP5;1 is essential for efficient boron uptake and plant development under boron
limitation. Plant Cell, 18 (6)
256 Functional analysis of metal transporters in Thlaspi caerulescens
Sangita Talukdar, Mark Aarts
Wageningen University
Heavy metal hyperaccumulation in plants is a poorly understood phenomenon. Transmembrane metal transporters
are assumed to play a key role in this process. In our research Zn transporters of Thlaspi caerulescens, a heavy metal
hyperaccumulator plant, are studied and compared to orthologues of Arabidopsis thaliana, a non-hyperaccumulator
plant. The ZTP1 Zn transporter gene shows 85% sequence similarity in the coding region with the ZAT/MTP1 gene of
Arabidopsis and is assumed to be localized in the vacuolar membrane and suggested to function in vacuolar metal loading.
This gene belongs to the Cation Diffusion Facilitator (CDF) family. The ZNT1 and ZNT2 genes are members of the Zrt,
Irt (ZIP)-like gene family and show 89% & 87% similarity with the ZIP4 and IRT3 genes of A. thaliana, respectively.
Constitutively high expression of ZNT1 and ZNT2 in roots, irrespective of the Zn concentration in the medium whereas
the A. thaliana ZIP4 and IRT3 genes are induced exclusively by Zn-deficiency, suggests a role for these genes in Zn
uptake. The proteins are assumed to be localized in the plasma membrane, conferring zinc uptake into the cytoplasm.
35S promoter -ZNT1 and -ZNT2 showed higher sensitivity and early flowering when grown on low Zn, than wild type
Columbia. 35S promoter - ZTP1 transgenic plants shows higher tolerance to high Zn compared to wild type. The response
of these genes to different concentrations of divalent metal ions (Zn, Cd, Mn, Fe and Cu) and the complementation of A.
thaliana mutants by T. caerulescens genes will be checked. Preliminary studies of transiently expressed ZNT1-GFP and
ZNT2-GFP constructs in cowpea protoplasts indicated localization in the plasma membrane. The regulation of expression
of these genes is studied in comparison to the orthologous genes in A. thaliana. In order to examine the response of the
ZIP4 promoter to different Zn media, transformed Arabidopsis plants with a ZIP4 promoter::GUS construct were studied,
which shows induction by low Zn only. The ZNT1 promoter was isolated by PCR using forward primers designed on
the A. thaliana gene upstream of ZIP4 and a reverse primer on the T. caerulescens ZNT1 cDNA (of which the upstream
gene is unknown). The promoters of ZIP4 were isolated in a similar method from Arabidopsis halleri, Arabidopsis lyrata
and the related Cochlearia pyrenaica. The expression of the ZIP4 genes of all these five species in response to different
Zn concentrations, will be studied by quantitative RT-PCR.