15th International Conference on Arabidopsis Research - TAIR

T07-001
The At4g12720 gene encoding a homologue of the
human GFG protein is active on ADP-ribose and
flavine adenine dinucleotide (FAD).
Olejnik Kamil(1, 1), Kraszewska Elzbieta(1, 1)
1-Plant Biochemistry Department, Institute of Biochemistry and Biophysics, PAS, Warsaw,Poland
Human GFG, a 35-kDa protein, is encoded by antisense fibroblast growth
factor (FGF-2) mRNA. The translation product of sense FGF-2 RNA is a member
of a growth factor family with mitogenic and hormone-regulatory functions.
Human antisense FGF-2 RNA, complementary to the 3’- untranslated
region of its sense partner, is involved in FGF-2 mRNA editing and stability.
However, apart from this function the antisense RNA encodes the GFG protein
which has homology to the MutT family of antimutator nucleotide hydrolases
and can partially complement this function in a MutT- deficient E. coli strain.
In the human pituitary the GFG protein enhances prolactine expression and
inhibits cell proliferation, independent of FGF-2 expression (1).
In the genome of Arabidopsis thaliana seven genes coding for proteins with
substantial homology to human GFG are indicated in the TIGR database.
Similarly to the human protein, they all possess, within their primary amino
acid sequences, conserved MutT/Nudix domains GX5EX7REUXEEXGU (U =Ile,
Leu or Val). This motif is characteristic for a widespread family of Nudix proteins
which catalyze mostly the hydrolysis of nucleoside diphosphates linked
to certain moieties X. The list of substrates of the Nudix enzymes includes:
nucleotide triphosphates NTP, nucleotide sugars, NADH, NAD, FAD, coenzymeA,
m7GTP-mRNA cap, dinucleoside polyphosphates and a non nucleoside
compounds - diphosphoinositol polyphosphates (2).
Three cDNA clones, coding for different Arabidopsis thaliana GFG proteins
(AtGFG), obtained from the Arabidopsis Biological Resource Center, Ohio,
USA, were subcloned into expression vectors. The first clone investigated,
At4g12720* (AtGFG1), was used for complementation tests in a E.coli mutT
mutator strain. We did not observe any complementation of MutT function
nor hydrolysis of mutagenic 8-oxoGTP, a main substrate of the MutT protein.
We have overexpressed AtGFG1 in E.coli and purified the recombinant
AtGFG1 protein. We confirmed the protein identity and integrity by Western
hybridization and mass spectrometry. The enzymatic activity was tested
with purified recombinant protein and a number of different substrates. At
reaction conditions typical for Nudix enzymes, at pH 8. 5 and in the presence
of Mg2+ ions, AtGFG1 was active mainly on ADP-ribose and FAD.
* the gene marked according to its position on chromosome, follow the TIGR
database
1.Asa, S.L. at al (2001) Molecular Endocrinology, 4, 589-599
2.Bessman, M.J. at al (1996) J.Biol.Chem. 271, 25059-2506
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin
T07-002
Characterisation of the transparent testa 10
mutant affected in Arabidopsis seed coat flavonoid
metabolism
Lucille Pourcel(1), Jean-Marc Routaboul(1), Michel Caboche(1), Loïc Lepiniec(1),
Isabelle Debeaujon(1)
1-Laboratoire de Biologie des Semences, INRA-INAPG, Institut Jean-Pierre Bourgin, Route de
St-Cyr, F-78026 Versailles Cedex, France
Flavonoids found in seeds have important impacts on agronomic seed qualities.
Particularly, they increase seed dormancy and longevity. Genetic and
molecular analysis of flavonoid metabolism in Arabidopsis thaliana can be
performed using mutant lines affected in seed coat pigmentation (transparent
testa mutants, « tt mutants»). To date, 22 loci involved in flavonoid biosynthesis
have been identified in Arabidopsis.
Here, we present the characterisation of the tt10 mutant. Browning of the
tt10 seed coat is delayed compared to that of the wild-type seed coat,
suggesting that an enzyme involved in tannin metabolism may be affected in
this mutant. We identified five new independent mutants that appeared to be
allelic to the tt10-1 mutant previously isolated. The six tt10 alleles were used
to identify the TT10 gene. Preliminary analysis by semi-quantitative RT-PCR
showed that TT10 is essentially expressed in developping siliques. TT10
promoter activity has been studied using promoter : reporter (GUS and GFP)
fusions in transgenic plants. Promoter activity is detected first in the endothelium
at early stages of embryo morphogenesis and, later on, the expression
spreads to the outer cell layers of the integuments. LC-ESI-MS-MS analysis
of flavonoids in tt10 and wild-type seeds is currently in process. Details on
gene and mutant characterisation will be presented and discussed.
T07 Metabolism (Primary, Secondary, Cross-Talk and Short Distance Metabolite Transport)