20S proteasome and accumulation of oxidized and ubiquitinated proteins in maize leaves subjected to cadmium stress.
ABSTRACT In order to examine the possible involvement of the 20S proteasome in degradation of oxidized proteins, the effects of different cadmium concentrations on its activities, protein abundance and oxidation level were studied using maize (Zea mays L.) leaf segments. The accumulation of carbonylated and ubiquitinated proteins was also investigated. Treatment with 50 microM CdCl(2) increased both trypsin- and PGPH-like activities of the 20S proteasome. The incremental changes in 20S proteasome activities were probably caused by an increased level of 20S proteasome oxidation, with this being responsible for degradation of the oxidized proteins. When leaf segments were treated with 100 microM CdCl(2), the chymotrysin- and trypsin-like activities of the 20S proteasome also decreased, with a concomitant increase in accumulation of carbonylated and ubiquitinated proteins. With both Cd(2+) concentrations, the abundance of the 20S proteasome protein remained similar to the control experiments. These results provide evidence for the involvement of this proteolytic system in cadmium-stressed plants.
- SourceAvailable from: Abdelilah Chaoui[Show abstract] [Hide abstract]
ABSTRACT: The role of the ubiquitin (Ub)-proteasome pathway and some endo- and aminopeptidases (EPs and APs, respectively) was studied in cotyledons of germinating bean seeds (Phaseolus vulgaris L.). The Ub system appeared to be important both in the early (3 days) and late (9 days) phases of germination. In the presence of copper, an increase in protein carbonylation and a decrease in reduced -SH pool occurred, indicating protein damage. This was associated with an enhancement in accumulation of malondialdehyde, a major product of lipid peroxidation, and an increase in content of hydrogen peroxide (H2O2), showing oxidative stress generation. Moreover, copper induced inactivation of the Ub-proteasome (EC 3.4.25) pathway and inhibition of leucine and proline aminopeptidase activities (EC 126.96.36.199 and EC 188.8.131.52, respectively), thus limiting their role in modulating essential metabolic processes, such as the removal of regulatory and oxidatively-damaged proteins. By contrast, total trypsin and chymotrypsin-like activities (EC 184.108.40.206 and EC 220.127.116.11, respectively) increased after copper exposure, in parallel with a decrease in their inhibitor capacities (i.e. trypsin inhibitor and chymotrypsin inhibitor activity), suggesting that these endoproteases are part of the protective mechanisms against copper stress.Plant Physiology and Biochemistry 01/2014; · 2.35 Impact Factor
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ABSTRACT: Cadmium (Cd) is a non-essential heavy metal that may be toxic or even lethal to plants as it can be easily taken up by the roots and loaded into the xylem to the leaves. Using soybean roots (Glycine max L) DM 4800, we have analysed various parameters related to reactive oxygen metabolism (ROS) and nitric oxide (NO) during a six-day Cd exposure. A rise in H2 O2 and NO, and to a lesser extent O2 (.-) content was observed after 6h exposure with a concomitant increase in lipid peroxidation and carbonyl group content. Both oxidative markers were significantly reduced after 24h. A second, higher wave of O2 (.-) production was also observed after 72h of exposure followed by a reduction until the end of the treatment. NOX and GOX might be involved in the initial Cd-induced ROS production and it appears that other sources may also participate. The analysis of antioxidative enzymes showed an increase in glutathione-S-transferase activity and in transcript levels and activity of enzymes involved in the ascorbate-glutathione cycle and the NADPH generating enzymes. These results suggest that soybean is able to respond rapidly to oxidative stress imposed by Cd by improving the availability of NADPH necessary for the ascorbate-glutathione cycle.Plant Cell and Environment 01/2014; · 5.91 Impact Factor
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ABSTRACT: Cadmium (Cd) is non-essential heavy metal, which in excess, exhibits deleterious effects to the most of the organisms. Mobilization of defense mechanisms against this toxic agent requires rapid activation of signaling pathways. The article presents recent advances in the research concerning cadmium signal transduction in plants. New insights into the involvement of reactive oxygen species (ROS), nitric oxide (NO), plant growth regulators, and Cd-induced protein modifications are reviewed. Moreover, the role of recently recognized Cd-associated signal elements, including micro RNAs and several cis- and trans-acting elements is discussed.Frontiers in Plant Science 01/2014; 5:245. · 3.64 Impact Factor
20S proteasome and accumulation of oxidized and ubiquitinated
proteins in maize leaves subjected to cadmium stress
Liliana B. Pena, Laura A. Pasquini, Marı ´a L. Tomaro, Susana M. Gallego*
Departamento de Quı ´mica Biolo ´gica, Facultad de Farmacia y Bioquı ´mica, Universidad de Buenos Aires,
Junı ´n 956, Buenos Aires (C1113AAC), Argentina
Received 16 August 2006; received in revised form 8 February 2007
Available online 30 March 2007
In order to examine the possible involvement of the 20S proteasome in degradation of oxidized proteins, the effects of different cad-
mium concentrations on its activities, protein abundance and oxidation level were studied using maize (Zea mays L.) leaf segments. The
accumulation of carbonylated and ubiquitinated proteins was also investigated. Treatment with 50 lM CdCl2increased both trypsin-
and PGPH-like activities of the 20S proteasome. The incremental changes in 20S proteasome activities were probably caused by an
increased level of 20S proteasome oxidation, with this being responsible for degradation of the oxidized proteins. When leaf segments
were treated with 100 lM CdCl2, the chymotrysin- and trypsin-like activities of the 20S proteasome also decreased, with a concomitant
increase in accumulation of carbonylated and ubiquitinated proteins. With both Cd2+concentrations, the abundance of the 20S protea-
some protein remained similar to the control experiments. These results provide evidence for the involvement of this proteolytic system in
? 2007 Elsevier Ltd. All rights reserved.
Keywords: Zea mays L.; Maize; Graminaceae; Proteasome; Protein oxidation
Cadmium is a potent poison for all living cells. Although
it is redox inactive, it is a well established oxidative stressor
by depleting glutathione and protein-bound sulfhydryl
groups, which result in production of reactive oxygen spe-
cies (ROS) such as superoxide ion, hydroxyl radicals and
hydrogen peroxide (Stohs et al., 2001; Romero-Puertas
et al., 2004). In turn, the reactive oxygen species can cause
modifications to the amino acids of proteins (Dean et al.,
1997; Nystro ¨m, 2005). However, only a limited number
of oxidative protein changes, such as protein disulphides
or methionine sulfoxides, can be enzymatically repaired
in vivo. By contrast, the bulk of the oxidized proteins must
be degraded to prevent accumulation of the unfolded pro-
tein forms (Grune et al., 2004). The intracellular levels of
oxidized proteins thus reflect the balance between the rate
of protein oxidation and the rate of oxidized protein
The proteasome-ubiquitin system is the major proteo-
lytic pathway, in both the cytoplasm and nucleus of
eukaryotes (Callis and Vierstra, 2000; Vierstra, 2003). In
mammalian cells, besides its function as the proteolytic
core of the 26S complex, numerous studies have demon-
strated its key role in degradation of oxidatively modified
proteins. Interestingly, no ATP and ubiquitin requirement
has ever been reported for proteolysis of oxidized proteins
in various systems. Therefore, it is generally believed today
that the 20S proteasome core complex is sufficient alone for
degradation of oxidized proteins in the cell cytoplasm and
nucleus (Shringarpure et al., 2001; Grune et al., 2003). Fur-
0031-9422/$ - see front matter ? 2007 Elsevier Ltd. All rights reserved.
*Corresponding author. Present address: Junı ´n 956, 1oPiso, Buenos
Aires (C1113AAC), Argentina. Tel.: +54 11 49648237; fax: +54 11
E-mail address: email@example.com (S.M. Gallego).
Phytochemistry 68 (2007) 1139–1146
thermore, cells with a non-functional ubiquitination cas-
cade were still able to remove oxidized proteins efficiently
(Shringarpure et al., 2003).
The 20S proteasome is arranged as a cylindrical stack of
four heptameric rings with two inner rings of b-type sub-
units, and two outer rings of a-type subunits. Each b-ring
contains three different proteolytically active sites, which
are classified based on their specificity towards short syn-
thetic peptides as: chymotrypsin-like, trypsin-like, and
caspase-like (peptidylglutamyl-peptide hydrolyzing) (Ing-
vardsen and Veierskov, 2001; Orlowski and Wilk, 2000).
In plants, cadmium causes a decrease in glutathione, this
being related to generation of reactive oxygen species and
accumulation of oxidized proteins (Gallego et al., 2005;
Rella ´n-A´lvarez et al., 2006). While the modification of
non-specific proteases activities was already reported
(Palma et al., 2002), little is known about the effect of cad-
mium on the proteasome. This is specially relevant because
of the potential role of the 20S proteasome for degradation
of oxidized proteins. The aim of the present work was,
therefore, to investigate if increasing cadmium concentra-
tion affects the 20S proteasome activity by oxidative mod-
ification. We also studied the quantitative and qualitative
pattern of carbonylated and ubiquitinated proteins pro-
duced by the metal treatment in maize leaves.
2.1. Effect of cadmium in maize whole plants
There was a significant decline in glutathione content in
the leaves of maize plants (50% of the control value) with
both Cd2+concentrations used (Fig. 1). Although glutathi-
one content decreased with metal addition, a significant
increase in carbonyl group content (20% respect to control
values) was also observed at the highest cadmium concen-
tration tested (100 lM) (Fig. 1).
2.2. Effect of cadmium in leaf segments of maize plants
To exclude possible differences in metal absorption and
distribution rates, direct treatment of leaf segments were
also performed. Carbonyl group contents were determined
in leaf segments as a protein oxidative damage index.
Leaves treated with 50 lM Cd2+for 24 h did not show
increased levels of carbonylated proteins, whereas treat-
ment with 100 lM Cd2+did (19% compared to control
plants) (Table 1).
Furthermore, while simultaneous treatment with 50 lM
Cd2+and MG132, a well known proteasome inhibitor,
resulted in a significant on accumulation of oxidized pro-
teins (20% increase), these values were not different from
that observed with 100 lM Cd2+alone (Table 1). Thus,
the addition of MG132 in the incubation medium did not
increase levels of oxidized protein with respect to control
The densitometric scanning of the immunodetection of
carbonyl residues was in agreement with that obtained by
spectrophotometric analysis. On the other hand, the qual-
itative pattern of carbonylated proteins (Fig. 2a) showed
that 100 lM Cd2+increased oxidation of proteins with a
molecular weight lower than 45 kDa, while SDS–PAGE
staining with Coomassie Brilliant Blue R-250 (Fig. 2b)
had similar protein profiles for control and Cd-treated leaf
2.3. Effect of cadmium on the ubiquitin-proteasome
Analysis of the ubiquitin-proteasome proteolytic system
in maize leaf segments was performed following cadmium
treatment for 24 h. Proteasome peptidase activities were
assayed by monitoring cleavage of three different fluoro-
genic peptide substrates: AAF-AMC, Boc-LSYR-AMC
and Clz-LLE-bNA for chymotrypsin-, trypsin-, and
PGPH-like activities, respectively, in either the absence
or presence of MG132, with results expressed as differ-
ences between both measurements (Fig. 3). When leaf
segments were treated with 50 lM Cd2+, trypsin- and
C 50 µM
nmol eq GSH g-1 FW
nmol C=O mg-1 protein
Fig. 1. Effect of cadmium on glutathione content (white) and protein
oxidation level (gray). Leaves of maize (Zea mays L.) plants were treated
with 50 and 100 lM CdCl2for 24 h in hydroponic culture. Values are
means ± SEM. Significant differences**P < 0.01 and***P < 0.001, accord-
ing to Tukey’s multiple range test.
Effect of cadmium on total carbonyl group content
Treatmentnmol C@O mg?1protein
50 lM CdCl2
50 lM CdCl2+ MG132
100 lM CdCl2
51.6 ± 1.9
52.1 ± 1.5
49.5 ± 2.2
62.0 ± 1.1a
61.3 ± 0.8a
Maize plants (Zea mays L.) were germinated 20 d and then leaf segments
were floated for 24 h in water either devoid of cadmium or containing 50
and 100 lM CdCl2. When the effect of the proteasome inhibitors was
investigated, 50 lMG132 was added to the incubation medium. Values are
means ± SEM. Significant differences.
aP < 0.05 according Tukey’s multiple range test.
L.B. Pena et al. / Phytochemistry 68 (2007) 1139–1146
PGPH-like activities increased 7% and 67%, respectively,
whereas chymotrypsin-like activity remained similar to
control. For the 100 lM Cd2+treatment, the chymotryp-
sin and trypsin-like activities decreased by about 82%
and 88% with respect to control, whereas the PGPH-like
activity remained essentially the same. Reduction in all
peptidase activities of the 20S proteasome were detected
when concentration higher than 100 lM cadmium were
used (data not shown).
Fig. 4 shows that the free ubiquitin and polyubiquiti-
nated protein contents remained similar to the controls in
maize leaf segments treated with 50 lM Cd2+. With
100 lM Cd2+treatment, however, together with an inhibi-
tion of proteasome activity, a small increase in the accumu-
lation of ubiquitin-conjugated proteins was observed (25%
respect to control).
2.4. Effect of cadmium on proteasome protein abundance and
To test whether modification of 20S proteasome activi-
ties in maize leaf segments, as induced by cadmium treat-
ment, were due to either decomposition of the quaternary
structure of the multimeric enzyme complex or to modifica-
tion of amino acids, a series of non-denaturing PAGE and
SDS–PAGE were performed. Western blots of a native 6%
(w/v) PAGE showed a single protein band (Fig. 5a). How-
ever, the abundance of the 20S proteasome protein content
in leaf segments, treated with Cd2+, remained similar to the
controls. Oxidation of the 20S complex induced by cad-
mium was also estimated using SDS–PAGE (12.5% w/v)
(Fig. 5b). It was observed that both Cd2+concentrations
generated a significant level of oxidation of its subunits,
with 100 lM resulting in more oxidation than 50 lM
(Fig. 5b). SDS–PAGE analysis of the 20S proteasome
before immunoprecipitation with anti-DNP also demon-
strated that the antibody (anti 20S proteasome) cross-
reacted with the 20S protein derivatized with 2,4 DNPH
and that the individual proteasome subunits were undam-
aged by cadmium.
50 µM 100 µM
Fig. 2. Effect of cadmium on patterns of total and oxidized proteins. (a)
Western blotting with anti-DNP antibody. (b) Coomassie Brilliant Blue R-
250 proteins staining. Maize plants (Zea mays L.) were germinated 20 d
with leaf segments then floated for 24 h in water either devoid of cadmium
or containing 50 and 100 lM CdCl2. Leaf extracts (50 lg total protein)
were subjected to SDS–PAGE (12% w/v polyacrylamide), with bands
visualized as described in Section 5. Gels and membranes were photo-
graphed with a Fotodyn and analyzed with GelPro software. The
positions of molecular mass markers (in kDa) are shown on the right.
Electrophoresis and western blot data shown are representative of three
blots with a total of four to five samples/group between the three blots.
Relative hydrolysis activity
Fig. 3. Effect of cadmium on leaf proteasome activity. Maize plants (Zea
mays L.) were treated as above (in Fig. 2). Trypsin-like, chymotrypsin-like
and peptidyl glutamyl peptide hydrolase (PGPH) proteasome activities
were measured in leaf extracts using three peptide substrates (Boc-LSYR-
AMC, AAF-AMC and Clz-LLE-bNA, respectively) of the 20S protea-
some in the absence or presence of the proteasome inhibitor MG132.
Enzymatic activities were normalized for protein concentrations and
expressed as percentages of activity present in the controls. Data represent
mean values ± SEM. Significant differences
***P < 0.001 were observed between control and treated plants according
to Tukey’s multiple range test.
*P < 0.05,
**P < 0.01 and
L.B. Pena et al. / Phytochemistry 68 (2007) 1139–1146
In maize plants growth inhibition, nutrient uptake
reduction, disturbances in the plant–water relationships
and induction of defense mechanisms, including increased
levels of several antioxidant enzymes, were previously
reported as consequence of cadmium stress (Pa ´l et al.,
Several studies have suggested that oxidative stress is
involved in Cd2+toxicity. This can occur by either inducing
oxygen free radical production or by decreasing levels of
enzymatic and non-enzymatic antioxidants (Benavides
et al., 2005), thereby resulting in protein carbonylation
(Romero-Puertas et al., 2002). In the study described
herein, however, using maize leaves, although cadmium
treatment decreased glutathione content, only the highest
concentration assayed (100 lM) resulted in oxidized pro-
tein formation. Berlett and Stadtman (1997) indicated that
protein carbonylation is an irreversible oxidative process
leading to a loss of function and to an increased susceptibil-
ity to proteolytic attack. However, very severely oxidized
proteins form large aggregates due to increased levels of
cross-linked hydrophobic, ionic, and covalent bonds, and
C 50 µM 100 µM
Fig. 4. Effect of cadmium on polyubiquitinated proteins accumulation.
Maize plants (Zea mays L.) were treated as in Fig. 2. After 24 h of
treatment, leaf extracts (50 lg total protein) were subjected to SDS–PAGE
(10% polyacrylamide, w/v). Western blotting was performed using the
anti-ubiquitin antibody and bands were visualized as described in the
Experimental. Ub = free ubiquitin, Ub-P = ubiquitinated proteins. Bands
were photographed with a Fotodyn, analyzed with GelPro software and
expressed in arbitrary units (assuming control value equal to 10 U), based
on absolute integrated optical density (IOD) of each band and line.
Significant differences***P < 0.001 according to Tukey’s multiple range
test. Molecular masses of standard proteins (in kDa) are indicated on the
right. The western blot data shown are representative of three blots with a
total of four to five samples/group between the three blots.
C 50 µM 100 µM
C50 µM100 µM
50 µM100 µM
C 50 µM 100 µM
Fig. 5. Effect of cadmium on 20S proteasome protein and identification of
20S proteasome oxidized protein. (a) Western blotting of native PAGE
(6% w/v). (b) Western blotting of SDS–PAGE (12.5% w/v) after
derivatization with 2,4 DNPH followed by immunoseparation with anti-
DNP. (c) Western blotting of SDS–PAGE (12.5% w/v) after derivatization
with 2,4 DNPH. Maize plants (Zea mays L.) were germinated 20 d and
then leaf segments were floated for 24 h in water devoid of cadmium or
containing 50 and 100 lM CdCl2. Western blotting was performed using
the anti-20S maize proteasome antibody and bands were visualized as
described in Section 5. Bands were photographed with a Fotodyn,
analyzed with GelPro software and expressed in arbitrary units (assuming
control value equal to 10 U), based on absolute integrated optical density
(IOD) of each band. Values are means ± SEM. The western blot data
shown are representative of three blots with a total of four to five samples/
group between the three blots.
L.B. Pena et al. / Phytochemistry 68 (2007) 1139–1146
thus become progressively more resistant to proteolysis
Incubation of maize leaf segments with 50 lM Cd2+
together with the proteasome inhibitor MG132 resulted
in accumulation of oxidized proteins. This indicate that,
as in mammalian cells, the recognition of hydrophobic
amino acid residues of oxidized proteins and their subse-
quent degradation by the 20S proteasome is a selective
mechanism to remove oxidatively damaged proteins from
the cytoplasm and nucleus, while proteases have a similar
role in organelles.
Fluorogenic kinetic assays using fluorogenic substrates
are extensively used to estimate proteasome activities in
mammalian and plant extracts. Although the substrates
are not proteasome specific, the difference observed in the
presence or absence of MG132 permitted estimation of
modifications in proteasome activities. AAF-AMC is fre-
quently used as substrate to asses in plants the activity of
the tripeptidyl peptidase 2 (TPP2), which is insensitive to
MG132 (Book et al., 2005). Although the peptide aldehyde
proteasome inhibitor MG132 inhibits some calpain type
protease, Kim et al. (2003) found that most of the hydro-
lytic activity in vitro indeed comes from the proteasome.
While maize leaf segments exposed to 50 lM cadmium
increased the 20S proteasome activity, neither variation
observed. Despite protein abundance remained unchanged,
an increase in the 20S proteasome oxidation was observed.
As suggested by Shringarpure et al. (2001), a number of
chemical modifications, most of which act to relax the
structure of the proteasome, can activate its proteolytic
activity. This mechanism seems to be similar to that
described in maize roots by sugar starvation (Basset
et al., 2002). On the other hand a decline of 26S protea-
some activity has been reported to occur after severe oxida-
tive stress produced by hydrogen peroxide, but no decline
in the activity of the 20S proteasome after moderate oxida-
tive stress has been detected (Reinheckel et al., 1998).
Under our experimental conditions, the moderate level
of oxidation produced by 50 lM cadmium may have
caused a slight dissociation of the complex and could have
been responsible for the increment in its activity, thus
avoiding accumulation of carbonylated proteins. However,
higher concentrations seem to result in partial inactivation
of the peptidic activities, similar to what was observed
under strong oxidative stress conditions in proteasome iso-
lated from human erythrocytes treated with increasing
H2O2concentrations (Reinheckel et al., 1998).
Conformational changes brought about by severe oxida-
tive modifications of specific amino acids in the 20S multi-
catalytic protein subunits may have resulted in inactivation
of the enzyme, with a concomitant increment in the oxi-
dized proteins. Cadmium sensitivity increased after a single
amino acid substitution in the b5-subunit of neuronal cells
(Li et al., 2004), while in yeast Ni2+resistance was achieved
by insertion of a maize cDNA encoding the a-subunit of
20S proteasome (Forzani et al., 2002). These findings
reinforce the notion that a proteasome deficiency unequiv-
ocally potentiates the harmful effects of oxidative stress
produced by cadmium in maize leaves.
Proteins extracted from control and cadmium treated
leaf segments showed similar patterns, at least within the
limits of SDS–PAGE and Coomassie staining techniques.
However, immunoblots with ubiquitin antibody revealed
an accumulation of ubiquitinated proteins (Ub-P) after
100 lM Cd2+treatment, thus suggesting the proteasome
inhibition. Proteasome dysfunction triggered by harmful
conditions such as oxidative stress (Okuda et al., 1997;
Lee et al., 2001) or aging (Keller et al., 2002) is likely to
decrease ubiquitinated protein degradation rate, causing
Ub-P accumulation. Likewise, it is known that plant expo-
sure to heavy metal ions induces changes in the ubiquitin-
dependent pathway. For example, Genschik et al. (1992)
reported that HgCl2treatment increased the expression of
ubiquitin genes in Nicotiana sylvestris, and a strong accu-
mulation of a ubiquitin conjugating enzyme (Ubc1) was
also observed in tomato after exposure to CdCl2(Feussner
et al., 1997). Other types of abiotic stress like darkness, UV
radiation, starvation and enhanced ozone level also influ-
enced polyubiquitin gene expression (Ingvardsen and
Taken together, our findings suggest that there is a
threshold response of the proteasome system to cadmium
stress mediated through oxidative modification of the pro-
teasome itself, which prevents accumulation of oxidatively
damaged protein in the cell. Under low cadmium treat-
ment, the increase in the 20S proteasome activity due to
its moderate oxidation was responsible for preventing oxi-
dized proteins accumulation. Higher cadmium concentra-
tiondecreased 20S proteasome
concomitant accumulation of oxidized and ubiquitinated
5.1. Plant material and growing conditions
Seeds of maize (Zea mays L., cv DK-682) were germi-
nated and grown in vermiculite in a controlled climate
room at 24 ± 2 ?C and 50% relative humidity, with a pho-
toperiod of 16 h light (intensity, 300 lmol m?2s?1). Ten
days old plants were removed from the pots, the roots were
then carefully and gently washed and transferred to sepa-
rate containers (3 L) for hydroponic culture, 20 plants
per container. In the whole plant assay, the hydroponic
nutrient solution was either half strength Hoagland’s nutri-
ent solution (Hoagland and Arnon, 1950), for the controls
or contained 50 and 100 lM of CdCl2, respectively. The
medium was continuously aerated. After 24 h treatment,
L.B. Pena et al. / Phytochemistry 68 (2007) 1139–1146
leaves were isolated and used for the determinations. For
leaf segment assays, leaf segments from 20 d plants (1 cm,
0.3 g) were floated for 24 h in glass flasks with H2O
(50 mL) (control), or containing 50 and 100 lM CdCl2.
When the effect of proteasome inhibitors was investigated,
MG132 (50 lM) was added to the incubation medium.
After 24 h, leaf segments were washed with distilled H2O
and analyzed as describe below. All experiments were
repeated three times with five replicates per treatment.
5.2. Glutathione assay
Non-protein thiols were extracted by homogenizing
leaves (1 g) in 0.1 N HCl (10 mL, pH 2) containing polyvi-
nylpyrrolidone (PVP) (1 g). After centrifugation at 10,000g
for 10 min at 4 ?C, each supernatant was used for analysis.
Total glutathione (reduced GSH plus oxidized GSSG) lev-
els were determined spectrophotometrically at 412 nm in
the homogenates using yeast glutathione reductase, 5,50
dithio-bis-(2-nitrobenzoic acid) (DTNB) and NADPH,
respectively (Anderson, 1985).
5.3. Carbonyl group determination
Extracts were prepared from leaf tissue (1 g), homoge-
nized in extraction buffer (3 mL) consisting of 100 mM
phosphate buffer (pH 7.4), 120 mM KCl and 1 mM EDTA
as follows. Each homogenate was centrifuged (10,000g for
20 min) with the supernatant fraction used for assays. Pro-
tein oxidation was measured in term of total carbonyl
group content by reaction with 2,4-dinitrophenylhydrazine
(Levine et al., 1990). Extracts (50 lg protein) that were pre-
viously derivatized with 2,4 DNPH were separated by
SDS–PAGE using 12% (w/v) running and 4% (w/v) stack-
ing polyacrylamide gels, respectively (Laemmli, 1970). Two
gels were run simultaneously: one for protein staining with
Coomassie Brilliant Blue R-250 and the other for immuno-
detection. Derivatized proteins were transferred onto nitro-
cellulose membranes and were detected with rabbit anti-
DNP primary antibody from Sigma–Aldrich (St Luis,
USA). Bands corresponding to oxidized proteins were visu-
alized by secondary goat anti-rabbit immunoglobulins con-
diaminobenzidine (DAB) as substrate.
5.4. Proteasome activity determinations
Chymotrypsin-like, trypsin-like, and peptidylglutamyl-
peptide hydrolase (PGPH) activities of the proteasome
were assessed in leaf extracts using synthetic peptide sub-
strates linked to the fluorescence reporters Ala-Ala-Phe-7-
amido-4-methylcoumarin (AAF-AMC), Boc-Leu-Ser-Thr-
Arg-7-amido-4-methylcoumarin (Boc-LSYR-AMC) and
the absence or presence of the proteasome inhibitor carbo-
benzoxy-Leu-Leu-leucinal (MG132) (Kim et al., 2003).
Leaf extracts (1 g) were prepared in 135 mM Tris–acetate
buffer (5 mL, pH 7.5), containing 12.5 mM KCl, 80 lM
EGTA, 6.25 mM 2-mercaptoethanol, and 0.17% (w/v)
octyl-b-D-glucopyranoside, respectively. Homogenized leaf
extracts were centrifuged with supernatants used for enzy-
matic activity determinations. Each reaction was started by
adding 100 mM HEPES–HCl buffer (100 lL, pH 7.5) and
synthetic substrate (2 lL stock solution at 50 lM in
dimethyl sulfoxide (DMSO)) to extract (100 lL). MG132
was added to the assay mixture at a final concentration
of 100 lM. Incubations were carried out at 37 ?C for 1 h.
Reactions were stopped by adding 220 mM sodium acetate
buffer (100 lL, pH 5.5) for chymotrypsin-like and trypsin-
like activities, and EtOH (300 lL) for PGHP activity. After
dilution in distilled H2O, AMC and NA radicals released
were measured fluorometrically (excitation 370 nm/emis-
sion 430 nm and excitation 333 nm/emission 450 nm,
respectively). Enzymatic activities were normalized for pro-
tein concentrations and expressed as percentages of activity
present in control extracts.
5.5. Western blot of 20S proteasome and polyubiquitinated
Samples (50 lg protein) were subjected to electropho-
retic analysis using 6% (w/v) native-polyacrylamide gel
(PAGE) and 12.5% (w/v) SDS–PAGE for the proteasome,
or 10% (w/v) SDS–PAGE for the polyubiquitinated pro-
teins. Gels were transferred onto polyvinylidene difluoride
membranes. Polyclonal antibodies, raised against the maize
20S proteasome (generously provided by Dr Brouquisse)
and ubiquitin-protein conjugates (Santa Cruz Biotechnol-
ogy Inc., Santa Cruz, CA) were also employed. Bands were
subsequently visualized using a secondary goat antibody
conjugated with horseradish peroxidase and stained using
3,30-diaminobenzidine (DAB) as substrate. Membranes
were photographed with a Fotodyn, analyzed with GelPro
software and expressed as arbitrary units (assuming control
value equal to 10 U), based on absolute integrated optical
density of each band for 20S proteasome or each line for
5.6. Immunoprecipitation and immunochemical detection of
proteasome carbonyl groups
Proteins (100 lg) derivatized with DNPH as mentioned
above, were separated by affinity chromatography. Anti-
bodies anti-DNP (50 lL) were linked to cyanogen bromide
activated Sepharose 4% agarose matrix (100 mg) from
Sigma–Aldrich (St Luis, USA). Samples were incubated
overnight at 4 ?C with an excess of anti-DNP-agarose resin
and then centrifuged for 5 min at 10,000g. Resin beads
were washed 3 times with Tris-buffered saline (TBS), with
pellets re-suspended in 100 mM glycine–HCl (50 lL, pH
2.5). After centrifugation, the pellets were discarded, with
the pH of the supernatants adjusted to 6.8 with 0.5 M
Tris–HCl buffer (5 lL, pH 8.8) and used for immunodetec-
tion of the 20S proteasome. DNPH derivatized proteins
L.B. Pena et al. / Phytochemistry 68 (2007) 1139–1146
were separated by 12.5% (w/v) SDS–PAGE. After electro-
transfer of the proteins to nitrocellulose membranes, the
proteasome was detected using maize anti-20S proteasome
primary antibodies and goat anti-rabbit immunoglobulins-
horseradish peroxidase conjugate, with DAB as substrate,
respectively. Membranes were photographed with a Foto-
dyn equipment, analyzed with GelPro software.
5.7. Protein determination
Protein concentrations were determined according to
Bradford (1976) using bovine serum albumin as standard.
Values are expressed as mean ± SEM. Differences
among treatments were analyzed by 1-way ANOVA, tak-
ing P < 0.05 as significant according to Tukey’s multiple
This work was supported by Grants from the Universi-
dad de Buenos Aires (Argentina) and from Consejo Nac-
ional de Investigaciones
(CONICET) (Argentina). L.A.P., M.L.T. and S.M.G. are
career investigators from CONICET. The authors thank
Dr R. Brouquisse (UMR Physiologie Cellulaire Ve ´ge ´tale,
Grenoble, France) for his generous donation of the anti-
body against maize 20S proteasome. We thank Myriam
S. Zawoznik for critical reading of the manuscript and
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