15th International Conference on Arabidopsis Research - TAIR

T07-053
Soluble Cytosolic Heteroglycans Acts as Substrate
for the Cytosolic (Pho 2) Phosphorylase
Fettke, Joerg(1), Tiessen, Axel(2), Eckermann, Nora(1), Steup, Martin(1)
1-Department of Plant Physiology, Institute of Biochemistry and Biology, University of Potsdam,
Karl-Liebknecht-Str. 24-25, Buildung 20, D-14476 Potsdam-Golm, Germany
2-Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Potsdam-Golm,
Germany
The subcellular distribution of starch-related enzymes and the phenotype
of several Arabidopsis mutants impaired in starch mobilization suggest that
the plastidial starch degradation is linked to a complex cytosolic glycan
metabolism. In this communication, a soluble heteroglycan (SHG) preparation
from leaves of Arabidopsis thaliana L. has been studied. The SHG, whose
major constituents are galactose, arabinose, and glucose, comprises several
glycans. Using membrane filtration, the SHG can be separated into a 10 kDa (SHGL) fraction. As revealed by field-flow-fractionation
(FFF), the latter can be resolved into two subfractions, designated
as I and II. Subcellular location of the various glycans was determined by
non-aqueous fractionation of leaf material. The various non-aqueous fractions
obtained were analysed by HPAEC-PAD and by FFF-DRI. All soluble glycans
are located outside the chloroplasts. The low molecular weight glycans
(SHGS) possess the same distribution as cytosolic marker proteins, such as
Pho 2 and DPE 2. The two glycans of SHGL exhibited an unequal distribution:
Subfraction I cofractionated with the cytosolic markers whereas subfraction
II did not. Thus, both SHGS and subfraction I of SHGL are cytosolic glycans
whereas subfraction II resides outside the cytosol. In in vitro assays subfraction
I acted as glucosyl acceptor for the cytosolic (Pho 2) phosphorylase
whereas subfraction II did not. Both subfractions possess a similar monomer
pattern that, due to the high proportion of arabinose and galactose, somehow
resembles arabinogalactans. However, the soluble glycans, especially SHGS
and subfraction I, possess a more complex structure, as they possess a
variety of minor compounds, such as glucosyl, mannosyl, xylosyl, rhamnosyl,
and fucosyl residues. In Arabidopsis mutants that are defective in distinct
starch-related enzymes both SHGS and subfraction I are affected whereas
subfraction II remains unchanged. As an example: In the Arabidopsis mutant
defective in the plastidial phosphoglucomutase (pPGM) the cytosolic glycans
differ from these of the wild type in the monomer composition and the molar
mass distribution. Interestingly, the pPGM mutant possesses a twofold higher
Pho 2 activity.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin
T07-054
Two interacting high-affinity sulfate transporters
regulate the uptake of sulfate in response to sulfur
conditions.
Naoko Yoshimoto(1), Kazuki Saito(2), Tomoyuki Yamaya(1), Hideki Takahashi(1)
1-RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan
2-Graduation School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku,
Chiba, 263-8522, Japan
SULTR1;1 and SULTR1;2 are the two high-affinity sulfate transporters
responsible for the initial sulfate acquisition in Arabidopsis roots. Studies on
transgenic plants expressing the fusion gene constructs of their promoter
region and green fluorescent protein demonstrated that SULTR1;1 and
SULTR1;2 co-localize at epidermal and cortical cells of roots. The inducibility
of their expression was different in response to the sulfur status; SULTR1;1
mRNA was strongly up-regulated by sulfur limitation in parallel with the increase
in the sulfate uptake capacity of roots, whereas the abundant isoform,
SULTR1;2, was less responsive to the changes in sulfur conditions. Contribution
of SULTR1;1 and SULTR1;2 to the total sulfate uptake was investigated
by the comparative analysis of sultr1;1, sultr1;2 and sultr1;1 sultr1;2 double
knockout. The uptake of sulfate decreased both in sultr1;1 and sultr1;2
single mutants under low-sulfate conditions. The sultr1;1 sultr1;2 double
knockout failed to absorb sulfate from µM concentration, and showed severe
growth defects. The actual sulfate uptake capacity measured in the wild-type
plants was significantly higher than the total contribution of SULTR1;1 and
SULTR1;2, which was estimated from the sum of sulfate influx in sultr1;1
and sultr1;2 single mutants, suggesting that SULTR1;1 and SULTR1;2 may
synergistically function in maximizing the sulfate uptake capacity under sulfur
limited conditions. Co-expression of SULTR1;1 and SULTR1;2 in yeast system
also resulted in increasing the sulfate influx rate under sulfur-deficient
conditions; however, this multiplying effect was abolished by the addition of
organic sulfur. These results strongly suggest the interplay of SULTR1;1 and
SULTR1;2 transporters as an essential regulatory mechanism that controls
sulfate uptake capacity in response to sulfur conditions fluctuating at the root
surface.
T07 Metabolism (Primary, Secondary, Cross-Talk and Short Distance Metabolite Transport)