Plant Science 149 1999 13 – 22
Leaf age- and paraquat concentration-dependent effects on the levels of enzymes protecting against photooxidative stress
Leonardo M. Casano , Mercedes Martı´n, Jose´ M. Zapata, Bartolome´ Sabater
Departamento de Biologı´a Vegetal, Uni6ersidad de Alcala´, Alcala´ de Henares,
28871
Madrid, Spain Received 30 April 1999; received in revised form 23 June 1999; accepted 12 July 1999
Abstract
Antioxidant protective enzymes are usually induced in leaves under conditions of increased active oxygen generation, such as high light intensity, low CO
2
fixation rate or in the presence of paraquat, which transports electrons from photosynthetic machinery to oxygen to form O
2
−
. However, at high photooxidative stress, even protective enzymes can be destroyed and leaf cells become dead. The protective role of several chloroplastic activities was evaluated at increasing photooxidative stress in barley
leaves of different ages. We investigated the effects of different paraquat concentrations combined with low and high light intensities in expanding and aged-senescent leaves on the activity of plastid peroxidase and on the activity and protein levels of
plastid superoxide dismutase SOD, glutathione reductase GR and NADH dehydrogenase of the complex including polypep- tides encoded by plastid ndh genes. The chloroplastic GR was the most sensitive to inactivation when photooxidative stress
increased. SOD was preferentially induced in young-expanding leaves while NADH dehydrogenase and peroxidase were preferentially induced in adult-senescent leaves. The results suggest a limited role of GR in the protection against photooxidative
stress and a close relation between the actions of Ndh complex and peroxidase. © 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Barley; Chloroplasts; Glutathione reductase; Hordeum 6ulgare; ndh genes; Peroxidase; Superoxide dismutase www.elsevier.comlocateplantsci
1. Introduction
Photooxidative stress arises in photosynthetic tissues when the rate of the production of reducing
power is higher than the rate of its re-oxidation, mainly by CO
2
reduction; e.g. in the light at low temperature or with low CO
2
supply when drought stress closes stomata [1 – 5]. In these con-
ditions, high concentrations of the reduced forms of photosynthetic electron carriers favour one-
electron transfer reaction to oxygen producing superoxide anion radical O
2
−
from which derives other active oxygen species, as hydrogen
peroxide H
2
O
2
and hydroxyl radical OH. Ac- tive oxygen species have deleterious effects in
many cellular functions since they react with un- saturated fatty acid components of membrane
lipids, proteins and nucleic acids. To overcome in part these problems, living cells have developed a
complex enzymic machinery which destroys O
2
−
and H
2
O
2
. Superoxide dismutases SOD [2,3,6], peroxidases [4,5,7], chloroplast Ndh complex [8,9]
the complex with NADH dehydrogenase activity which includes polypeptides encoded by plastid
ndh genes [10,11] and glutathione reductases GR [12,13], among other enzymic activities, have been
proposed to be involved in reactions which scav- enge O
2
−
andor H
2
O
2
. Usually, enzymes involved in O
2
−
and H
2
O
2
scavenging in chloroplasts increase their levels when light intensity quickly increases or in the
presence of toxic compounds as paraquat [14] which captures electrons from photosynthetic elec-
tron transport to form O
2
−
from oxygen. The increase of protective enzymes under photooxida-
Corresponding author. Tel.: + 34-1-885-4911; fax: + 34-1-885- 5066.
E-mail address
:
bvlcobioveg.alcala.es L.M. Casano 0168-945299 - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 3 8 - 7
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
