15th International Conference on Arabidopsis Research - TAIR

T07-017
A biotechnological approach to reduce
photorespiratory losses. Effects of the expression of
Glycolate oxidase in plastids of Arabidopsis thaliana.
Verónica Maurino(1), Holger Fahnenstich(1), Ulf-Ingo Flügge(1)
1-Department of Botany, University of Cologne, Gyrhofstr. 15, 50931, Cologne, Germany.
The oxygenase reaction catalyzed by RubisCO is the first reaction of the
C2-photosynthetic carbon cycle. The prime function of the C2-pathway is to
salvage glycolate-2-P by conversion to glycerate-3-P, which re-enters the
C3-reductive cycle. In C3-plants, both CO2 and NH3 are released within
the mitochondria, resulting in a loss of at least 25% of the CO2 fixed in
ambient air. In this way, photorespiration is considered to be an energy
wasting process. But, it is also involved in the prevention of photooxidation
and the regeneration of metabolites. We attempt to introduce two alternative
glycolate catabolic cycles into chloroplasts of A. thaliana in order to create an
autoregulatory cycle, which results in an attenuation of the photorespiratory
pathway. In both cycles, the first step is catalyzed by glycolate oxidase (GOX).
Arabidopsis thaliana plants were transformed with a construct harboring an
endogenous GOX-cDNA cloned downstream of a plastidic transit peptide
and the CaMV35S-promoter. Selected homozygous lines were analyzed
by Southern blot for the number of transgene insertions. GOX plants are
characterized by retarded growth and yellowish leaves during the first
weeks. RT-PCR analyses showed no co-suppression of the endogenous GOX
expression. By week 5, GOX activity and the levels of glyoxylate and H2O2
were increased in the transformants, accompanied by decreasing photosynthetic
efficiencies. By week 7, the transformants resemble the WT, showing
the same H2O2 level as the WT, and also the electron transport rate tends
to approach the WT level. The GOX phenotype can be reverted by growing
the plants under high CO2 or by co-expressing catalase in the plastids. Coexpression
of tartronate-semialdehyde reductase in the plastids did not revert
the GOX phenotype. Here, we present the strategies used to characterize
the transgenic plants overexpressing GOX in plastids and conclude that GOX
plants are suitable for further engineering of the C2-photosynthetic carbon
cycle.
Tolbert, N. (1997) The C2 oxidative photosynthetic carbon cycle. Annu. Rev. Plant Physiol. Plant
Mol. Biol. 48: 1-25.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin
T07-018
A gene family in Arabidopsis with cystine-lyase and
tyrosine aminotransferase-activities
Heike Hollaender-Czytko(1), Janine Grabowski(1), Iris Sandorf(1), Katrin
Weckermann(1), Elmar W. Weiler(1)
1-Lehrstuhl Pflanzenphysiologie, Ruhr-Universitaet Bochum, 44801 Bochum, Germany
In the genome of Arabidopsis thaliana seven genes are annotated to be
tyrosine aminotransferase(TAT)-like proteins with sequence similarities between
43 and 78 %. While the function of some of these genes has not been
elucidated so far, for At4g23600 (CORI3), which is coronatine-inducible (1),
the main activity was shown to be a cystine lyase-activity (2). For At2g20610,
a participation as a C-S lyase in the glucosinolate metabolism has been
demonstrated (3) and for At5g52970, tyrosine aminotransferase-activity
could be measured. Both enzymes use pyridoxal phosphate as a cofactor
and participate in amino acid metabolism. Characterization of heterologously
expressed CORI3 shows that the cystine lyase has a strong preference for
the substrate cystine while djenkolic acid, S-methyl- and S-ethyl-cysteine
are used to a lesser extent. The enzyme has a broad pH- and temperatureoptimum.
Expression studies applying semi-quantitative RT-PCR indicate that,
while some members of the family are not or hardly transcribed, inducibility
with coronatine, octadecanoids and herbicides for the others varies drastically.
Total specific tyrosine aminotransferase-activity in Arabidopsis rises
twofold upon activation with coronatine, while total specific cystine lyaseactivity
increases by a factor of 15 ¯18. Expression of the genes in different
organs of the plant show divergent patterns. Transgenic plants have been
made using a plasmid with the 35-S promotor, BASTA-resistance and the
complete cDNA sequence of CORI3. Characterization of these plants showing
the expression of CORI3 on the RNA level using semiquantitive RT-PCR and
on the enzymatic level will be presented.
1:Lopukhina et al,Plant Physiol 126,1678(2001); 2: Jones et al,JBC278,10291(2002); 3:Mikkelsen
et al,Plant J 37,70(2004)
T07 Metabolism (Primary, Secondary, Cross-Talk and Short Distance Metabolite Transport)