Bariloche protein symposium argentine society for biochemistry and molecular biology

42
BIOCELL, 27 (Suppl. I), 2003
PL-C3.
ENHANCED TOLERANCE TO IRON DEFICIENCY IN
TRANSGENIC TOBACCO PLANTS EXPRESSING A
BACTERIAL FLAVODOXIN
Tognetti, Vanesa B.
1
; Zurbriggen, M.D.
1
; Morandi, E.
2
; Fillat,
M.
3
; Valle, Estela M.
1
 and Carrillo, Nestor
1
.
1
IBR, Fac.Cs. Bqcas y Farm., UNR, Rosario, Argentina. 
2
Fac. Cs
Agrarias, UNR, Rosario, Argentina. 
3
Fac. de Ciencias, Zaragoza,
España. E-mail: [e-mail]
One of the most severe limitations for agricultural development is
the availability of iron, particularly pronounced in plants grown
in alkaline, calcareous soils, which cover more than one third of
earth’s surface and represent a major deterrent for agriculture.
Soil-based organisms have evolved various adaptive mechanisms
to ameliorate the consequences of iron deficit. For instance,
cyanobacteria and phytoplankton induce the expression of
flavodoxin (Fld) to replace ferredoxins (Fd), whose iron-sulphur
clusters cannot be assembled in iron-restricted media. Fld are small
hydrophillic proteins containing FMN as prosthetic group that
collaborate with and/or substitute Fd in many different processes,
including photosynthesis. They are found in prokaryotes and some
algae, but not in vascular plants. The reported recruitment of
bacterial Fld under iron deficit encouraged us to assess the
behaviour of tobacco plants expressing Fld in chloroplasts (pfld)
or cytosol (cfld) when exposed to low iron concentrations. We
report here that transgenic tobacco lines expressing Anabaena
PCC7119 Fld in chloroplasts displayed enhanced resistance
relative to wild-type or cfld plants to low iron availability.
Tolerance results from functional replacement of Fd. Modulation
of Fld levels in plants might therefore provide entirely novel tools
to improve the field tolerance of agronomically important crops.
PL-C4.
INDUCTION OF TWO ENDOPROTEOLYTIC ACTIVITIES
IN SENESCENT WHEAT LEAVES
Roberts, Irma N.; Passeron, Susana and Barneix, Atilio J.
IBYF-CONICET, Buenos Aires, Argentina. E-mail:
[e-mail]
Senescence is the ultimate stage of every plant organ. One of its
most characteristic features is the high rate of protein degradation.
Endoproteolytic activity measured as azocasein hydrolysis is
strongly increased in dark-induced senescent wheat leaves.
Analysis of extracts from senescent leaves on DEAE-Sepharose
allowed separation of the azocaseinolytic activity in two major
peaks. One appeared in the flow-through (P
1
) and the other one
(P
2
) retained in the column was eluted by ionic strength. Previous
isolation and purification of P
1
 activity allowed its identification
as a subtilisin-like serine endoprotease. An antiserum prepared
against purified P
1
 was used for western blot analysis. It showed
that this enzyme appeared in senescent leaves induced by
incubation in darkness but was not detectable when induction was
achieved by nitrogen deprivation. A band of 84 kDa in SDS-PAGE
correlating with P
2 
activity was also revealed through several steps
of purification. This protein, already present in non-senescent
leaves increased its amount in extracts from senescent leaves
induced by darkness or by nitrogen starvation. The fact that P
2
reacted with the antibody against the subtilisin-like endoprotease
P
1
, indicates that these two enzymes are structurally related. As
previously demonstrated for P
1
, P
2
 activity was inhibited by the
protease inhibitors chymostatin and PMSF and was able to
hydrolyse Suc-AAPFpNA suggesting that P
2
 also belongs to the
chymotrypsin-like proteases.
PL-C6.
PECTINOLYTIC ACTIVITIES IN POTATO-FUSARIUM
INTERACTION
Olivieri, Florencia P.; Machinandiarena, Milagros F. and Daleo
Gustavo R.
Instituto de Investigaciones Biológicas, Mar del Plata, Argentina.
E-mail: [e-mail]
To colonize potato tissue, Fusarium solani f.sp. eumartii degrades
plant cell wall. The aim of this work is to relate the pectinolytic
activities (Polygalacturonases, PG; Pectin Methyl esterases, PME)
with the different susceptibility to Fusarium of two potato cultivars:
Spunta, moderately resistant and Pampeana, susceptible. In
addition, we compared two F. solani isolates: F. solani  f.sp.
eumartii isolate 3122, pathogenic and F. solani isolate 1402, no
pathogenic. Both isolates grown in liquid culture containing pectin
as carbon source, produced similar amount of PG activity.
Conversely, in potato intercellular washing fluids (IWF), this
activity was higher for the susceptible cultivar inoculated with
pathogenic isolate than for the no pathogenic one. Also the PG
isoforms detected by zymograms were different. The amount of
total PG activity in the IWF changed in each cultivar when Ca++
was present in the reaction mixture. When 0.1 mM CaCl2 was
added, PG activity of infected Spunta IWF was higher than in
absence of CaCl2. For Pampeana IWF, the effect of Ca++ was
less evident. On the other hand, we compared the PME activity
between Spunta and Pampeana cultivars. Different isoforms were
visualized by zymograms. We suggest that the different PME
activity between both cultivars could modify the PG activity
produced by the pathogenic fungus. In addition, preliminary
evidences indicate that the major level of Polygalacturonase-
inhibiting proteins accumulated in the more resistant cultivar could
be related with its low level of PG activity.
PL-C7.
INVOLVEMENT OF LeCDPK AND PP2A IN RESPONSE
TO ABIOTIC STRESS IN TOMATO PLANTS
Daniela Capiati, Silvia Marina Pais, María Paula Coluccio,
María T. Téllez-Iñón.
INGEBI-CONICET, FCEyN, UBA. Vuelta de Obligado 2490, 2do.
piso. (1428) Buenos Aires. Argentina. E-mail:
[e-mail]
Plants have developed different mechanisms to avoid damage cused
by exposure to different stress conditions as biotic (virus, fungus,
hervibores) and abiotic (cold, drought, salinity) factors. The
defensive response involves the activation of signal transduction
pathways that lead to the modulation of gene expression. A calcium-
dependent protein kinase, CDPK, induced local and systemically
by mechanical damage in tomato (Lycopersicon esculentum) plants,
was described previously in our lab. To obtain more information
about the involvement of this kinase in plant defense response,
LeCDPK expression was determined in tomato plants exposed to
different abiotic stresses. The kinase was induced by high salinity
and cold in different parts of the plant. Protein phosphatase PP2A
expression was also determined suggesting its particiaption in the
response to abiotic stress. To determine if the systemic induction
of LeCDPK is mediated by systemin, the expression of the kinase
was determined in response to systemin. The results obtained
indicate that LeCDPK is induced in response to high salinity and
cold, being the systemical induction mediated by systemin, and
also suggest the involvement of the phosphatase PP2A in the
response to abiotic stress.

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