tive stress is currently accepted as indicative of their role in the scavenging of O
2
−
andor H
2
O
2
. However, sometimes, strong enough photooxida-
tive stress masks the induction of protective en- zymes because high concentrations of active
oxygen species quickly degrade the same enzymes which scavenge them. One possibility is that differ-
ent scavenging systems are functional at different stress intensities and at different developmental
stages of the leaves. Thus, to deduce the involve- ment of a given enzyme in protection against
photooxidative stress a range of stress intensity should be assayed in leaves at different develop-
mental stages.
In this work we have subjected young-expand- ing primary leaves 7-day-old and aged-senescent
primary leaves 14-day-old of barley to different photooxidative stress intensities produced by two
light intensities and a range of paraquat concen- trations. We have investigated the effects of these
treatments on the levels of chloroplastic SOD, hydroquinol peroxidase, GR and Ndh complex
activities and on the levels of SOD, GR and Ndh complex protein, to determine the involvement of
these activities in the protection of chloroplasts against photooxidative stress and the inactivation
of the same activities by far strong photooxidative stress.
2. Materials and methods
2
.
1
. Plant materials Barley Hordeum 6ulgare L cv. Hassan was
grown on vermiculite in a controlled growth chamber at 23°C under a 16 h photoperiod of 100
m mol photon m
− 2
s
− 1
white light as described [3]. In the present work, we have used primary leaves
of 7- and 14-day-old plants as young expanding and aged-senescent leaves respectively. Ten sub-
apical leaf segments 3 cm length were cut after 4 – 5 h from the beginning of photoperiod, and
incubated at 23°C during 20 h with 10 ml of different concentrations of paraquat in growing
light GL 100 mmol photon m
− 2
s
− 1
or relative photooxidative light PhL 300 mmol photon m
− 2
s
− 1
. In this experimental system, wounding pro- vokes a transient increase of some antioxidant
enzymes between 2 and 6 h after incubation. Thereafter, they stabilise at a level which depends
on the incubation conditions [3].
2
.
2
. Preparation of leaf crude extracts For zymographic and Western blot assays, ac-
tivities and proteins were assayed in whole leaf extracts obtained as follows: ten 3-cm leaf seg-
ments were homogenised with a mortar and pestle in 2 ml of 50 mM potassium phosphate pH 7.01
mM
L
-ascorbic acid1 mM EDTA5 wv polyvinylpirrolidone, and centrifuged at 500 × g
for 10 min. Except for extracts for the assay of GR and SOD, Triton X-100 was added to super-
natant to make a final 2 wv solution and gently stirred for 30 min. The suspension was
centrifuged at 20 000 × g for 30 min. Supernatants contained 0.7 – 1.3 mg protein per ml.
2
.
3
. Isolation of chloroplasts Enzymes corresponding to the specific chloro-
plastic activities investigated in crude extracts were identified by comparison with those of chloroplas-
tic fractions and by parallel zymograms and West- ern blots after native PAGE see 2.4.
Intact chloroplasts were isolated as described [8]. Leaf segments were homogenised in an Omni-
mixer Sorvall with six volumes of freshly pre- pared isotonic buffer E 50 mM potassium
phosphate1 mM
L
-ascorbic acid1 mM EDTA 0.33 M sorbitol, pH 7.0 supplemented with 5
wv polyvinylpyrrolidone. The homogenate was filtered through eight layers of muslin and cen-
trifuged at 200 × g for 5 min. The supernatant was centrifuged at 2000 × g for 10 min. The pellet of
chloroplasts was washed with buffer E 10 ml per 1 g original leaves to obtain a preparation of
chloroplasts free from soluble or mitochondrial fractions [7 – 9]. To obtain high percentages \
80 of intact chloroplasts, all steps from leaf segments to pellet of washed chloroplasts were
performed at 0 – 5°C in no more than 45 min. The chloroplast pellet was resuspended for osmotic
shock in buffer E without sorbitol buffer H at 0 – 4°C with gentle shaking during 6 min and then
centrifuged for 15 min at 4500 × g. The superna- tant was used to assay SOD and GR. The thy-
lakoid pellet was resuspended to approximately 2 mg protein per ml in buffer H and used for
solubilisation of thylakoid-bound Ndh complex or peroxidase.
The membranous fraction was washed with Tri- ton X-100 0.2 mg detergent per mg protein and
gently stirred for 15 min. After centrifugation at 10 000 × g for 20 min, the Ndh complex was solu-
bilised from the pellet, which was resuspended in 20 mM Tris – HCl pH 7.05 mM EDTA, by adding
10 wv Triton X-100 to make a final concentra- tion of 2 5 mg detergent per mg protein and
gently stirred for 30 min. The suspension was centrifuged at 105 000 × g for 45 min. The super-
natant around 4 mg protein per ml, ca. 40 of original thylakoid proteins was the NADH dehy-
drogenase solubilised fraction.
Peroxidase was solubilised from the thylakoid membranous fraction by adding 10 wv Triton
X-100 to make a final 1.5 solution and gently stirred for 45 min. The suspension was centrifuged
at 20 000 × g for 30 min. The supernatant around 0.9 mg protein per ml contained the peroxidase
solubilised from thylakoid.
All the isolation procedures Sections 2.2 and 2.3 were performed at 4°C.
2
.
4
. Gel electrophoresis, immunoassays and zymograms
Native PAGE was carried out at 5°C usually with 20 – 100 mg protein samples in a linear gradi-
ent gel of 3 – 10 polyacrylamide 2.5 bis-acry- lamide in the same way as SDS-PAGE with the
exception that gels contained 0.1 wv Triton X-100 for NADH dehydrogenase and peroxidaxe
zymograms instead of SDS [15] or neither SDS nor Triton X-100 for SOD and GR zymograms.
For immunoblot analyses, after SDS-PAGE, proteins were transferred to PVDF membranes
Millipore. Immunocomplexes with antibodies prepared against the NDH-F polypeptide encoded
by the ndhF gene [9], CuZn SOD chloroplastic [16], or chloroplastic GR [13] were detected with
the peroxidase Western blotting analysis system Boehringer.
For zymograms, NADH dehydrogenase activity of Ndh complex was detected by incubation of gel
slices for 20 – 30 min at 30°C in darkness in 50 mM potassium phosphate pH 8.01 mM Na
2
– EDTA 0.2 mM NADH and 0.5 mg per ml nitro blue
tetrazolium. In controls without NADH no stain developed. Staining for peroxidase was performed
by following standard methods with 4-methoxy-a- naphthol [7] as substrate. SOD was detected in gel
by the photochemical nitro blue tetrazolium stain- ing method [17] and CuZn SOD identified as
before [16]. GR was detected by incubating with NADPH, GSSG and 3-4,5-dimethylthiazol-2-yl-
2,5-diphenyltetrazolium bromide [12]. Diaphorase bands were discriminated by performing the stain-
ing procedure without GSSG. Bands were scanned with a UVP Easy Digital Image analyser to com-
paratively quantify activity values which were ex- pressed as percentages of the reference activity
that of freshly detached primary leaves of 7- or 14-day-old plants.
2
.
5
. Enzyme assays As reference for absolute rates of some activities
see legends for Figs. 3 and 5, the activity NADH:FeCN oxidoreductase specific of Ndh
complex was assayed at 30°C by measuring the reduction of FeCN at 420 nm extinction coeffi-
cient: 1.03 mM
− 1
cm
− 1
and the oxidation of NADH at 340 nm extinction coefficient: 6.22
mM
− 1
cm
− 1
in a Beckman DU-650 spectropho- tometer. The reaction mixture, with a final volume
of 1.0 ml, included 50 mM potassium phosphate pH 7.51 mM Na
2
– EDTA0.2 mM NADH1 mM FeCN and variable enzyme preparations. The
rate was determined from linear absorbance de- crease between 45 and 240 s. Control values, ob-
tained without protein, were subtracted. No detectable transformation of one substrate was
observed in controls without the other substrate.
The spectrophotometric assay of peroxidase was performed at 30°C in a 1.0 ml assay volume
containing 0.5 mM hydroquinone HQ0.1 mM H
2
O
2
50 mM potassium phosphate buffer pH 7.0. After mixing, the enzymatic reaction was initiated
by adding 10 ml of enzyme. The oxidation of HQ was recorded as the increase in absorbance at 250
nm extinction coefficient: 19 mM
− 1
cm
− 1
over a time period of 2 min. The absorbance increases
were always linear with respect to time. Appropri- ate controls were subtracted [7].
Specific activities are expressed as mmol of NADH or HQ consumed per min per mg protein.
2
.
6
. Other determinations Protein concentration was quantified by the
method of Bradford [18] with a Protein Assay Kit Bio-Rad using bovine serum albumin as a stan-
dard. Chlorophylls were determined according to Wathley and Arnon [19] and carotenoids accord-
ing to Lichtenthaler [20].
All reported results were reproduced at least three times. When appropriate, standard devia-
tions are indicated by bars in figures.
3. Results
