Bariloche protein symposium argentine society for biochemistry and molecular biology

142
BIOCELL, 27 (Suppl. I), 2003
PL-P34.
HELIANTHUS ANNUUS L. RESPONSES TO CADMIUM
STRESS. ANTIOXIDANTS AS MARKERS OF THE METAL
TOXICITY
Groppa, María D.; Benavides, María P. and Tomaro, María L.
Dpto. de Química Biológica, Facultad de Farmacia y Bioquímica,
Universidad de Buenos Aires, Argentina. E-mail:
[e-mail]
Cadmium is an important environmental pollutant present in soils,
toxic to living cells and strongly phytotoxic. Cadmium toxicity
was evaluated in relation to antioxidant parameters such as
antioxidant enzymes, polyamines (Pas) and proline in sunflower
plants during 10 days of growth under 0.1 mM and 1 mM CdCl
2
.
Plant growth and root length were significantly inhibited by the
higher cadmium concentration from the third day. Except for
guaiacol peroxidase, all the antioxidant enzymes (catalase,
ascorbate peroxidase and glutathione reductase) showed a
significant increase under 1 mM CdCl
2
 from day 7 in shoots and
roots, though in roots the rise in the enzymes activity was lower.
Glutathione content also increased by day 7 in roots and shoots.
Polyamines and proline increased from day 7 in shoots and roots
under both cadmium concentrations. Thiobarbituric acid reactive
substances measured as a marker of oxidative damage increased
in roots and shoots only by 1 mM CdCl
2
. This increase was higher
in roots than in shoots. Cadmium stress induced by the highest
metal concentration produced an evident growth inhibition and
oxidative damage, in spite of the increment of all of the measured
parameters. The increase in polyamines and proline, postulated
as antioxidants, offered an additional protection. However, other
parameters, in addition to growth and oxidative damage, could
add information about the exact magnitude of the damage exerted
by the metal.
PL-P35.
BEHAVIOR OF PEROXIDASE ISOFORMS IN
SUNFLOWER PLANTS SUBJECTED TO UV-B
RADIATION
Yannarelli, Gustavo G.; Gallego, Susana M. and Tomaro, María L.
Dpto. de Química Biológica, Facultad de Farmacia y Bioquímica,
Universidad de Buenos Aires, Argentina. E-mail:
[e-mail]
Ozone depletion results in an increase in the biological harmful
solar ultraviolet-B radiation (280-320 nm) reaching the surface of
the earth. UV-B radiation has deleterious effects in plant growth
and fundamental physiological processes. Recent studies confirm
that hydrogen peroxide (H
2
O
2
) is a signaling molecule in plants
that mediates responses to abiotic and biotic stresses. Peroxidases
(POD) are heme proteins that catalyze the oxidation of a range of
substrates by H
2
O
2
, and are important in the H
2
O
2 
turnover. The
objective of this work was to determine the effect of UV-B radiation
and H
2
O
2
 on peroxidase isoforms as a potential mechanism of UV
tolerance in plants. Seven and fifteen day-old sunflower plants
were treated with UV-B doses 30 kJ/m
2
 or increased H
2
O
2
concentrations (0.5-100 µM). Peroxidase isozymes were analyzed
in cotyledons and leaves by native-PAGE and FPLC. In cotyledons
two POD isozymes were enhanced (pI: 5.0 and pI: 5.2) and
appeared a new isoform (pI: 5.4) in UV-B treated plants, compared
with control plants. Similar results were obtained with H
2
O
2
treatments. UV-B treatment induced pI: 5.0 and pI: 5.2 POD
isoforms in leaves. Our results are indicating that the UV-B
signaling in plants are mediated by H
2
O
2 
and POD plays and
important role in this response.
PL-P36.
ARABIDOPSIS ACTIVATION-TAGGED  MUTANTS
TOLERANT TO OXIDATIVE STRESS
Bondino, Hernán G.; Scarpeci, Telma E.; Carrillo, Néstor; Valle,
Estela M.
IBR (CONICET), Facultad de Ciencias Bioquímicas y
Farmacéuticas, UNR, Suipacha 531, S2002LRK Rosario,
Argentina. E-mail: [e-mail]
Plants have evolved a dynamic network of antioxidant defenses
that serve to reduce the oxidative damage caused by environmental
stress. To identify new components of this network, we had
performed large-scale gain-of-function screenings of Arabidopsis
mutants. These activation-tagged mutants were generated by
Weigel´s group, who constructed a vector (pSKI015) with four
copies of an enhancer element from the cauliflower mosaic virus
35S gene. Seedlings (8.850 lines; segregated into 86 pools) were
subjected to oxidative stress by spraying with the herbicide
Paraquat (methyl viologen, MV). MV generates superoxide by
leakage of electrons at photosystem I in chloroplasts under
illumination. Mutants more tolerant to oxidative stress were easily
identified in comparison with wild-type lines, which were not able
to survive. In a primary screening, 18 putative pools with higher
tolerance to MV were isolated. In a secondary screening, 11 lines
of mutants displaying strong tolerance to MV were confirmed.
Plasmid rescues were carried out to identify the segment of plant
DNA adjacent to the activation-tagged border, and the nearly gene
expressed, which could be responsible for the tolerant phenotype.
The function of the newly identify sequences in the antioxidant
defense network will be discussed.
PL-P37.
SCREENING A TOBACCO cDNA LIBRARY WITH
PURIFIED MAIZE NADP-ME ANTIBODIES AND
EXPRESSION OF THE PRODUCT OBTAINED IN E. COLI
Lara, María V.; Müller, Gabriela; Drincovich, María F. and
Andreo, Carlos S.
CEFOBI, Facultad de Ciencias Bioquímicas y Farmacéuticas
(UNR). Suipacha 531. Rosario. Argentina. E-mail:
[e-mail]
Previous studies indicated that different antibodies´ batches against
the purified photosynthetic NADP-malic enzyme (NADP-ME)
from maize leaves cross-react with a 72 kDa protein, detected in
different tissues of several plant species. A 72 kDa protein with
low NADP-ME activity was also purified from several plant
sources. Thus, this 72 kDa-protein was pointed out as a non-
photosynthetic NADP-ME. Nevertheless, we recently found that
the cDNA supposed to encode for this NADP-ME in maize roots,
codifies in fact for a novel highly active 66 kDa NADP-ME. In
this way, in order to identify the nature of the 72 kDa protein, we
used the maize purified NADP-ME antibodies to perform a
screening of a cDNA tobacco library; as Western blot analysis of
tobacco leaf protein with these antibodies detected only a 72 kDa-
signal. After the screening, we obtained 8 independent clones, 4
of which were completely sequenced. Blast analysis of the
sequence obtained indicated that it was 80 to 93% identical to
Hsp70 from different plant sources. Prediction analysis of the
sequence obtained (AY253326) showed that codifies for a possible
cytosolic 70,876 Da protein. The cDNA for this Hsp70 was
connected in frame to a pET32 expression vector to over-express
the protein in E. coli. The association between the recombinant
NADP-ME and Hsp70 proteins in vitro, suggest that such
association can be the reason for the cross-reaction of the anti-
purified maize NADP-ME antibodies.

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