All reported results were reproduced at least three times. When appropriate, standard devia-
tions are indicated by bars in figures.
3. Results
3
.
1
. Effects of photooxidati6e stress on the le6els of proteins, chlorophylls and carotenoids
Leaves of 7- and 14-day-old plants were used to know the influence of leaf age on the photooxida-
tive effects of paraquat and high light intensity. Until leaf detachment, barley plants were growing
in photoperiodic regime under the low intensity light 100 mmol m
− 2
s
− 1
. Fig. 1 shows the changes of soluble proteins, chlorophylls and
carotenoids of barley leaf segments after incuba- tion during 20 h under low light intensity growing
light, GL, 100 mmol m
− 2
s
− 1
and photooxidative light PhL, 300 mmol m
− 2
s
− 1
and 0, 50, 100 or 300 nM paraquat.
Paraquat produced a progressive yellowing of leaf segments which was dependent, at least, of its
concentration and on the light intensity. At a concentration of 100 nM, and even more at 300
nM, paraquat produced a clear photooxidative damage
when measured
by soluble
protein, chlorophyll or carotenoids decrease. The decreases
of proteins, chlorophylls and carotenoids were more pronounced when the paraquat incubations
were carried out under high PhL, empty symbols than under low GL, full symbols light intensities.
Paraquat at 50 nM did not have any apparent effects in comparison with incubation in the ab-
sence of paraquat. In addition, the photooxidative effect of paraquat stimulating the decrease of
proteins was more pronounced in 14-day-old aged leaves than in 7-day-old expanding leaves. Al-
though chlorophylls diminished slightly more in expanding than in aged leaves at concentrations
up to 100 nM paraquat, they decreased more in aged than in expanding leaves at 300 nM
paraquat. The paraquat-stimulated carotenoid de- crease, while more pronounced at high PhL than
at low GL light intensity, was rather similar in expanding and in aged leaves.
Apparently, under the conditions of assays, only concentrations of paraquat higher than 50 nM
increased the production of active oxygen species large enough to stimulate the degradation of
proteins, chlorophylls and carotenoids with re- spect to incubation in the absence of paraquat.
3
.
2
. Effects of photooxidati6e stress on chloroplast SOD acti6ity and protein
SOD has been thoroughly investigated as an activity typically induced under photooxidative
stress, presumably as a response to scavenge O
2
−
. Thus, it is a good tool to know how much strong
photooxidative conditions are required to surpass the induction of protective response by the delete-
rious effects of active oxygen species.
Fig. 1. Effects of leaf age, photooxidative light and paraquat concentration on the levels of proteins, chlorophylls and
carotenoid of leaf segments. Expanding 7-day-old and aged- senescent 14-day-old leaves of barley were incubated at 23°C
during 20 h with the indicated concentrations of paraquat PT in growing light GL 100 mmol photon m
− 2
s
− 1
or relative photooxidative light PhL 300 mmol photon m
− 2
s
− 1
. Remaining
soluble proteins,
chlorophylls and
carotenoids were expressed as percentages of the values in freshly detached leaves: 12.3 mg protein · g FW
− 1
, 975 mg chlorophyll · g FW
− 1
and 337 mg carotenoid · g FW
− 1
for 7-day-old plants and 11.1 mg protein · g FW
− 1
, 896 mg chlorophyll · g FW
− 1
and 302 mg carotenoid · g FW
− 1
for 14-day-old plants.
Fig. 2. Effects of leaf age, photooxidative light and paraquat concentration on the levels of CuZn SOD activity and
protein. A Typical zymogram of plastid CuZn SOD includ- ing activities of freshly detached leaves T
and of leaves incubated at 23°C during 20 h with the indicated concentra-
tions of paraquat PT in growing light GL or relative photooxidative light PhL. Twenty mg protein of leaf crude
extracts were loaded per lane. B Specific activities of CuZn SOD deduced from experiments as those of A. C CuZn
SOD protein level relative to total soluble protein in leaves treated as in A and deduced from Western blots not repre-
sented with antibody against CuZn SOD. Twenty mg protein were loaded per lane. In B and C, respectively, activities and
protein were expressed as percentages of the values in freshly detached 7-day-old leaves or 14-day-old plants for compari-
son see T
of A. Values are means of at least three different experiments.
aged 14-day-old leaves Fig. 2A, numerical data not represented and increased further after 20 h
incubation of leaves under GL 60 or PhL 80 Fig. 2A and B. When photooxidative
stress was increased by rising paraquat concentra- tion from 0 to 300 nM, SOD activity increased
almost linearly in expanding leaves under GL. At high light intensity PhL, SOD specific activity of
expanding leaves increased more than at GL up to 100 nM paraquat concentration. A further in-
crease of paraquat concentration 300 nM deter- mined a decrease of SOD activity, presumably
because the inductive effect of photooxidative stress on SOD was surpassed by the inactivation
and degradation of SOD produced by the high concentrations of active oxygen species.
The overall pattern of SOD protein levels in expanding leaves receiving different light and
paraquat treatments Fig. 2C was similar to that of SOD activity, although increases of activity in
respect to zero time were around twice of the corresponding increases of protein, which sug-
gested that photooxidative stress, produced by PhL, paraquat or both factors, stimulated the
synthesis of new SOD protein and favoured the activation or inhibited the inactivation of SOD
protein.
When 14-day-old leaves were cut and incubated under GL, SOD activity increased some 40 in
respect to zero time Fig. 2A. The presence of paraquat up to 100 nM did not cause a further
increase of SOD activity Fig. 2B. In addition, PhL and paraquat did not increase SOD activity
in these leaves. These results agree with previous findings [3] describing the lost of sensitivity to
SOD activity induction in senescent leaves. De- tachment and incubation under GL and PhL in-
creased SOD protein Fig. 2C more than SOD activity Fig. 2B; but, similarly to the effect on
activity, PhL incubation decreased SOD protein levels in respect to GL incubation. It is possible
that most of new synthesised SOD protein was inactive in aged leaves. Apparently reactive oxygen
species inactivated SOD to a form still reacting with polyclonal antibodies.
These results suggest that although photooxida- tive stress induced the synthesis of SOD protein, at
high photooxidative stress PhL and 300 nM paraquat for expanding leaves and lower paraquat
concentrations for senescent leaves SOD was in- activated at a higher rate than it was induced.
Chloroplastic SOD activity and protein was esti- mated from zymograms and Western blots per-
formed as described elsewhere [16]. Zero-time SOD specific activity in expanding
7-day-old leaves was about 65 higher than in
3
.
3
. Effects of photooxidati6e stress on chloroplast NADH dehydrogenase complex
Ndh acti6ity and
protein Zymograms comparison of crude extracts and
thylakoid solubilised complex and Western blot assays results not shown allowed identification of
NADH dehydrogenase of plastid Ndh complex as a well defined second low migration band in crude
extract which corresponded to the first low migrat- ing band in thylakoids.
Zero-time Ndh specific activity was about 80 higher in aged 14-day-old leaves than in expanding
7-day-old leaves Fig. 3A, numerical data not represented. The effects of increasing photooxida-
tive stress produced by low and high irradiances and different paraquat concentrations on Ndh
activity and protein detected with antibody against NDH-F Fig. 3 were rather similar to the
effects on SOD activity: at low – moderate pho- tooxidative stress in respect to the previous grow-
ing conditions activity and protein increased while at high photooxidative stress activity and
protein decreased. There were however some dif- ferences. While the induction of SOD activity was
mainly observed in expanding leaves compare
, with , in Fig. 2, the induction of Ndh activity and protein was more pronounced in aged
leaves and required milder photooxidative condi- tions in aged than in expanding leaves Fig. 3.
The induction of Ndh activity and protein and their inactivation by excessive photooxidative
stress, in general, required lower concentration of paraquat at high PhL open symbols than at
low GL filled symbols light intensity, indicating that high light intensities and paraquat acted by
similar compensatory effects: by increasing the concentration of reactive oxygen species. The re-
sults also supported a role for Ndh complex on the protection against reactive oxygen species.
3
.
4
. Effects of photooxidati6e stress on thylakoid peroxidase
Thylakoid peroxidase oxidised several quinones [7], among them plastoquinone, and has been pro-
posed to scavenge H
2
O
2
by oxidising the plas- toquinone reduced by the action of Ndh complex.
The activity of thylakoid peroxidase may be deter- mined in crude extract by zymographic analysis
because its low migration allows us to distinguish it from other peroxidases Fig. 4.
Zero-time thylakoid peroxidase specific activity was about 200 higher in aged 14-day-old leaves
than in expanding 7-day-old leaves Fig. 5A, numerical data not represented. Thylakoid perox-
idase increased in expanding leaves when photoox-
Fig. 3. Effects of leaf age, photooxidative light and paraquat concentration on the levels of Ndh activity and NDH-F
protein. A Typical zymogram of plastid NADH dehydroge- nase activity of the Ndh complex including activities of
freshly detached leaves T
and of leaves incubated at 23°C during 20 h with the indicated concentrations of paraquat
PT in growing light GL or relative photooxidative light PhL. One-hundred mg protein of leaf crude extracts were
loaded per lane. B Specific activities of Ndh complex de- duced from experiments as those of A. C Level of NDH-F
protein of Ndh complex relative to total soluble protein in leaves treated as in A and deduced from Western blots with
antibody against NDH-F. Twenty mg protein of leaf crude extracts were loaded per lane. In B and C, respectively,
activities and protein were expressed as percentages of the values in freshly detached 7-day-old leaves 4 nmol NADH
oxidised min
− 1
mg protein
− 1
or 14-day-old plants 7 nmol NADH oxidised min
− 1
mg protein
− 1
for comparison see T
of A. Values are means of at least three different experi- ments.
Fig. 4. Comparative isoenzyme pattern of whole leaf extract and thylakoid peroxidases after staining with 4-methoxy-a-
naphtol. Forty mg proteins was loaded per lane. Arrow indi- cates the migration of thylakoid peroxidase.
idative stress was raised by increasing paraquat concentrations Fig. 5. The inductive effect of
paraquat was particularly pronounced at high PhL relative light intensity and, although no
clear effect of inhibition by excess of paraquat was observed under the conditions assayed, at least,
the inductive effect was almost saturated between 100 and 300 nM paraquat.
As for Ndh activity Fig. 3, the increase of peroxidase activity Fig. 5 in response to pho-
tooxidative stress was more pronounced in aged than in expanding leaves. Very strong photooxida-
tive stress 300 nM paraquat inactivated peroxi- dase which reached the maximun at around 100
nM paraquat with both GL and PhL in aged leaves.
3
.
5
. Effects of photooxidati6e stress on glutathione reductase
GR acti6ity and protein
After non-denaturing electrophoresis and zy- mography, three main GR activities bands were
detected Fig. 6A: one low migrating extra- chloroplastic and a couple of fast migrating
chloroplastic results not shown. The three activi- ties reacted with our polyclonal antibodies [13]
and could not be distinguished by Western blot after SDS-PAGE. Thus, in Fig. 6 we represent the
effect of different photooxidative treatments on chloroplast GR activities in B and on total GR
protein in C.
In both type of leaves, chloroplastic GRs in- creased after 20 h incubation without paraquat in
GL and PhL, in respect to zero time. The presence of 50 nM paraquat further increased the activity
of leaves incubated under PhL. Paraquat concen- trations higher than 50 nM in PhL and any
paraquat concentration under GL decreased the chloroplastic activities Fig. 6B but, almost in a
correlative way, they increased the extrachloro- plastic GR activity Fig. 6A. Significantly,
paraquat concentration had little effect on the level of total GR protein Fig. 6C.
Although zero time chloroplastic GR specific activities in aged 14-day-old leaves was about 44
higher than in expanding 7-day-old leaves Fig. 6A, numerical data not represented, the in-
creases of chloroplastic GR activity at 0 under GL and PhL or 50 nM paraquat under PhL
Fig. 6B were more pronounced in expanding than in aged leaves.
Fig. 5. Effects of leaf age, photooxidative light and paraquat concentration on the levels of thylakoid peroxidase activity.
A Typical zymogram of thylakoid peroxidase including activities of freshly detached leaves T
and of leaves incu- bated at 23°C during 20 h with the indicated concentrations
of paraquat PT in growing light GL or relative photooxi- dative light PhL. Fifty mg protein of leaf crude extracts were
loaded per lane. B Specific thylakoid peroxidase activities deduced from at least three different experiments as those of
A. In B, activities were expressed as percentages of the values in freshly detached 7-day-old leaves 25 nmol HQ oxidised
min
− 1
mg protein
− 1
or 14-day-old plants 75 nmol HQ oxidised min
− 1
mg protein
− 1
for comparison see T of A.
4. Discussion
