15th International Conference on Arabidopsis Research - TAIR

T07-007
Functional analysis of cell wall components by
expressing a xyloglucanase in Arabidopsis thaliana
Carsten H. Hansen(1), Ulrike Hänsel(1), Kirk M. Schnorr(2), Markus Pauly(1)
1-Max Planck Institute of Molecular Plant Physiology, Golm, Am Mühlenberg 1, Germany
2-Novozymes, 1BM1.05 Novo Alle, 2880 Bagsvaerd, Denmark
Xyloglucan is a component of the plant cell wall and is believed to crosslink
cellulose microfibrils and thereby contribute to the strength of the wall.
Furthermore, xyloglucan metabolism seems to be involved in cell growth.
The aim of the project is to describe the effect of altering the xyloglucan
composition in plants on growth and development and on the physicochemical
properties of the cell wall.
A xyloglucanase (xyloglucan specific endo-1,4-ß-glucanase) from Aspergillus
aculeatus was transformed into A. thaliana using two different vector-systems
(pBinAR, pSRN). The pBinAR vector contains a CaMV 35S promoter, which
normally leads to high constitutive expression of the gene, whereas pSRN
has an ethanol inducible expression system. No A. thaliana transformants
were obtained when the xyloglucanase was expressed behind the CaMV 35S
promoter, suggesting that high expression of the xyloglucanase is lethal to
the plant. The xyloglucanase was successfully transformed into A. thaliana
using the pSRN vector. After treatment of the transformed plants with low
concentrations of ethanol, the xyloglucanase could be detected by Western
blot analysis. The expression level of the xyloglucanase had a direct correlation
with the amount of ethanol used for induction. Increased xyloglucanase
expression lead to inhibition of growth and formation of necrotic spots.
Further phenotypic parameters of the xyloglucanase expressing plants will be
presented and discussed.
T07 Metabolism (Primary, Secondary, Cross-Talk and Short Distance Metabolite Transport)
T07-008
A second starch phosphorylating enzyme is required
for normal starch degradation in Arabidopsis leaves:
the phosphoglucan, water dikinase (PWD)
Oliver Kötting(1), Axel Tiessen(2), Peter Geigenberger(2), Martin Steup(1), Gerhard
Ritte(1)
1-University of Potsdam, Institute for Biochemistry and Biology, Plant Physiology, Karl-Liebknecht-
Str. 24-25, Haus 20, 14476 Golm
2-Max-Planck-Institute of Molecular Plant Physiology, Am Mühlberg 1, 14476 Golm
Recent findings showed that degradability of transitory starch is strongly
influenced by its phosphorylation state. Until now only one starch phosphorylating
enzyme has been characterised, the a-glucan, water dikinase (GWD,
EC 2.7.9.4; formerly designated as R1) (1). In GWD-deficient sex1 mutants
of Arabidopsis starch breakdown is strongly impaired resulting in a starch
excess phenotype (2). However, the link between phosphorylation and degradation
of starch is not understood at present.
In order to identify enzymes whose activity depend on phosphate esters in
starch we searched for proteins with high affinity to phosphorylated starch.
In an in vitro binding assay we could identify a previously unknown protein,
preliminarily called OK1 (At5g26570; GenBank-Acc. AJ635427), which
exhibits a much higher interaction with phosphorylated starch compared with
non-phosphorylated starch. Interestingly, the OK1 sequence displays homology
to the GWD sequence. Similar to GWD, the recombinant OK1 protein
posseses starch phosphorylating activity. However, its activity strictly depends
on preceding phosphorylation of the starch substrate by GWD. Further
characterisation of the OK1 activity revealed that OK1 incorporates the bphosphate
of ATP into phosphoglucans, whereas the g-phosphate of ATP is
transferred to water. An autocatalytically phosphorylated OK1 protein occurs
as an intermediate of this reaction. Unlike GWD, which phosphorylates glucose
units of starch both at C-6 and C-3 positions at a ratio of about 2:1, OK1
links the phosphate predominantly to C-3. As revealed by cell fractionation
and subsequent immunoblotting using a polyclonal anti-OK1 antibody OK1 is
localised in the chloroplast. Transgenic Arabidopsis plants with reduced OK1
expression exhibit a starch excess phenotype.
From these data we conclude that OK1 is a chloroplastidic phosphoglucan,
water dikinase (PWD) which is required for normal degradation of leaf starch
in Arabidopsis.
(1) Ritte et al. (2002) PNAS 99, 7166-7171.
(2) Yu et al. (2001) Plant Cell 13, 1907-1918.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin