Bariloche protein symposium argentine society for biochemistry and molecular biology

146
BIOCELL, 27 (Suppl. I), 2003
PL-P50.
Arabidopsis thaliana TRANSGENIC PLANTS EXPRESSING
OAT ADC UNDER CONTROL OF RD29A PROMOTER
Alet AI, Sanchez DH, Ruiz OA.
IIB-INTECh (UNSAM-CONICET), Chascomús, Bs. As., Argentina.
E-mail: [e-mail]
Biotechnological uses of transgenic plants usually relays on
constitutive transgene expression. However, sometimes the
constitutive expression of transgenes might not be desirable. In
such cases, an alternatively technological approach could be use
of an inducible promoter. To evaluate this hypothesis, we developed
a binary vector encoding the oat–arginine decarboxylase gene
(ADC; EC 4.1.1.19), since this enzyme is known to be critical in
polyamine metabolism and can be considered the main supply of
putrescine in plants subjected to abiotic stresses. In general, the
function of the RD29A gene product is still unknown. It responds
to different signals produced by osmotic and cold stresses.
Dehydration and low temperature induce RD29A gene expression
showing a biphasic kinetic in the homologous system. It was
suggested that RD29A promoter has at least three kinds of cis
acting regulatory elements. One of them, denominated ABRE (ABA
Responding Element) is induced in ABA-dependent manner. In
our laboratory, homozygote transgenic plants of Arabidopsis
thaliana harboring this binary vector have been obtained, and they
didn’t show deleterious phenotypes frequently associated with
constitutive expression of ADC. The ADC induced activity by ABA
spraying was accompanied by changes in free polyamines levels.
Potential regulatory roles of the increased polyamines levels on
the induction of rd29A will be discussed.
PL-P51.
CHARACTERIZATION AND PREVENTION OF THE
PROTEOLYTIC CLEAVAGE OF PLANT PHOSPHO-
ENOLPYRUVATE CARBOXYKINASE
Martín M, and Podestá FE.
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI).
Suipacha 531. 2000 Rosario, Argentina. E-mail:
[e-mail]
In plants, phosphoenolpyruvate carboxykinase (PEPCK) catalyses
a key step in the gluconeogenesis during germination of fat storing
seeds, plays an important role in photosynthetic carbon assimilation
in some CAM and C
4
 plants, as well as in the CO
2
–concentrating
mechanism of certain algae. Plants PEPCKs have unique, species-
specific, N-terminal extensions that are rapidly lost by proteolysis
during preparation of cell extracts. Being the site of reversible
phosphorylation, proteolytic cleavage of these extensions hinders
the study of the regulation of PEPCK activity. The aim of this
study was to find proper conditions to extract and purify native
PEPCK from endosperm of 5 days old Ricinus communis
germinating seeds. PEPCK presented a subunit molecular mass
of 80 kDa in extracts obtained under denaturating conditions when
analyzed by western blot using affinity-purified antibodies against
truncated PEPCK from pineapple leaves. Studying the time course
state of PEPCK subunit in extracts made at different pH values
with and without DTT, we concluded that a cysteine endopeptidase
might be responsible for the degradation of PEPCK to a truncated
subunit form of 66 kDa. A wide range of protease inhibitors was
tested but none was completely useful to prevent proteolysis. A
combination of high pH and protease inhibitors was found to be
most effective to prevent proteolytic cleavage and maintain activity
of PEPCK.
PL-P52.
STUDY OF EXPRESSION OF H6h GENE IN BRUGMANSIA
CANDIDA HAIRY ROOT CULTURES
Marconi,  Patricia; Cardillo, Alejandra; Alvarez, Alejandra and
Giulietti, Ana M.
Cát. Micro. Ind. Y Biotecno., FFyB, UBA. Junin 954, Bs. As.,
C.P. 1113, Argentina. E-mail:  [e-mail]
The tropane alkaloids hyoscyamine and scopolamine are valuable
drugs employed as antispasmodics and in the treatment of motion
sickness. Their production by biotechnology processes is under
study in Brugmansia candida hairy roots (HR) cultures. Elicitation
is a strategy used to increase secondary metabolites including
alkaloids. In previous work, we reported that acetate buffer is able
to increase hyoscyamine (700%) and scopolamine (200%) in these
HR cultures. Hyoscyamine-6-
β-hydroxylase (H6H) is the enzyme
responsible for converts hyoscyamine into scopolamine. In order
to understand the difference levels of alkaloids reached after
elicitation in this work, we study the expression of H6h mRNA in
HR cultures elicited with acetate buffer. B. candida hairy root
cultures were obtained from seedlings transformed with
Agrobacterium rhizogenes LBA9402 according to Giulietti et al.
(1993). In elicitation experiments 0, 1.0 and 2.0 mM of acetate
buffer (Ac
-
) was added to the culture medium. The samples were
collected at 0, 6, 12 and 24 h after subculture. Total RNA was
obtained with RNeasy Plant Kit (Qiagen). cDNA synthesis was
done using Superscript II reverse Transcriptase (Life Technologies).
PCR reaction was done on bases of Hyoscyamus niger h6h gene
specific primer design due to high homology founded for the
transcript among Solanaceae family. The results obtained shown
the presence of H6hmRNA after 24 h of elicited treatment. Strong
expression of h6hgene was detected with 2.0 mM of Ac
-
 buffer
while a weak expression was detected at 1.0 mM of Ac
-
 buffer.
PL-P53.
ENHANCED OXIDATIVE STRESS TOLERANCE OF
TOBACCO TRANSGENIC PLANTS OVEREXPRESSING
BOTH FERREDOXIN-NADP(H) REDUCTASE AND
FLAVODOXIN
Lodeyro, Anabella; Rodriguez Virasoro, Ramiro; Poli, Hugo;
Tognetti, Vanesa; Valle, Estela M. and Carrillo, Néstor.
Instituto de Biología Molecular y Celular de Rosario (IBR,
CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas,
Universidad Nacional de Rosario, Suipacha 531, S2002LRK,
Rosario, Argentina. E-mail: [e-mail]
Ferredoxin-NADP(H) reductases (FNR) catalyse the reversible
electron transfer between two molecules of obligatory one-electron
carriers as ferredoxin and flavodoxin (Fd) and a single molecule
of NADP(H). Tobacco plants expressing Anabaena PCC7119
flavodoxin displayed enhanced tolerance to a variety of oxidative
stress conditions. Recently, plants overexpressing FNR in
chloroplasts were also obtained. In this work, we showed that this
tobacco plants overexpressing FNR were more tolerant to excess
light or to the reactive oxygen species generated by the herbicide
methyl viologen (MV). We also evaluated the tolerance to MV of
transgenic plants overexpressing both FNR and the cyanobacterial
flavodoxin. These plants were obtained either by cross-fertilization
or by transient expression of FNR directed to chloroplasts or cytosol
developed by leaf infiltration with Agrobacterium tumefasciens
carrying the appropriate plasmid construct. Results related to the
enhanced tolerance to MV of these double transgenic plants will
be discussed.

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