UDC 581.132:588.17
BIBLID 0021-3225 (1998) 34 p. 503-522
Review article
ACTIVE OXYGEN SPECIES AND PHOTOSYNTHESIS:
MEHLER AND ASCORBATE PEROXIDASE REACTIONS
Sonja VELJOVIû-JOVANOVIû*
Center for Multidisciplinary Studies of the Belgrade University, Yugoslavia
Veljoviü-Jovanoviü Sonja (1998): Active oxygen species and photo-synthesis:
Mehler and ascorbate peroxidase reactions.- Iugoslav. Physiol. Pharmacol.
Acta, Vol. 34, 503-522.
Oxygen is a natural acceptor of electrons from the photosynthetic
electron transport chain, during which the superoxide anion radical is formed
in the thylakoids. Rapid reduction of O2.- to H2 O, mediated by Cu Zn
superoxide dismutase and ascorbate peroxidase attached to the stromal
thylakoids, occurs before active oxygen species can diffuse to the stroma of
the chloroplasts. A continuos reduction of dehydroascorbate through the
ascorbate-glutathione cycle is performed in illuminated chloroplasts, with a
concomitant oxidation of NADPH+. As a result, scavenging of O2.- and H2 O2
in chloroplasts is achieved by utilizing the reducing power from photosynthetic electron transport. Such a system can be considered of benefit to
plants under conditions of decreased photosynthetic carbon reduction.
During photorespiration H2O2 is formed in peroxisomes (especially in
C3 chloroplasts) and scavenged by the catalase enclosed in these organelles.
The decrease in the catalase activity under various environmental stresses
Corresponding author: Sonja Veljoviü-Jovanoviü, Centar za multidiscipliarne studije,
29.Novembra 142, 11000 Beograd, Yugoslavija; fax (+381) 11 761 433;
e-mail: [email protected]
*
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IUGOSLAV.PHYSIOL.PHARMACOL.ACTA, Vol. 34, No.2, 503-522, 1998
could also contribute to the increase in the concentration of the cellular H2O2.
During the reduction of H2O2 monodehydroascorbate is formed, and it can
serve as an endogenous spin probe (detectable by EPR) in photooxidative
conditions. The results presented show that simultaneous measurement of
fluorescence, photosynthetic carbon assimilation and EPR spectroscopy can
be a valuable tool in studying photosynthetic systems under stress. In
conditions when the photosynthetic reduction of carbon is decreased, the
Mehler and the ascorbate peroxidase reactions are a possible alternative route
for the dissipation of surplus reducing power. A possible role of chloroplastic
ascorbate peroxidase and monodehydroascorbate reductase in scavenging
non-chloroplastic H2O2 is also proposed, and a role of this system in the
dissipation of excess light energy is discussed.
Key words: ascorbate peroxidase, EPR, monodehydroascorbate, photoinhibition, photorespiration, photosynthetic electron transport
INTRODUCTION
Plants under natural conditions are frequently exposed to an excess of electromagnetic radiation which cannot be completely utilized in photosynthesis. This excess
radiation can exhibit a damaging effect on the photosynthetic apparatus, and plants as
a whole. Various environmental stress factors which limit the CO2 uptake (water stress,
closure of stomata) or reduce the activities of the Calvin-Benson cycle enzymes
(pollutants, low temperatures, ultraviolet irradiation, growth reduction) in combination
with high light intensities, suppress photosynthetic CO2 assimilation and can lead to
photoinhibition (Kok, 1956; Powles,1984; Osmond, 1994). Under the same unfavorable conditions for CO2 assimilation, plants are also suffering from an oxidative stress
(Mishra and Singhal, 1992; Quartacci and Navaro-Izzo, 1992; Pastori and Tripi, 1993;
Polle and Rennenberg, 1993; Schittenhelm et al., 1993; Quartacci et al., 1994).
Besides, the involvement of activated oxygen species in promoting photoinhibition has
been indicated (Takahama and Nishimura, 1975; Aro et al., 1990; Richter et al., 1990;
Setlik et al.,1990).
Leaves are well equipped with numerous protective mechanisms involved in
preventing oxidative and photoinhibitory damage. The resulting negative effects on
plants depends on the capacity of cellular systems to scavenge activated oxygen species
and to prevent or to repair harmful effects of light on the photosynthetic electron
transport components. One of the first regulatory mechanisms that develops in an
excess of light is an increasing dissipation of excess excitation energy in PSII through
heat, causing non-photochemical quenching of PSII chlorophyll a fluorescence (Butler,
1978; Lavorel and Etienne, 1977; Krause and Weis, 1991). The increase in kD (rate
constant for thermal de-excitation of excited chlorophylls) depends on the proton
gradient across the thylakoid membrane (Krause 1973; Briantais et al., 1980) and
associated violaxanthin deepoxidation to zeaxanthin (Demmig-Adams, 1990). Thus,
the ûpH plays an important role in the regulation of photochemical activity of PSII
(Weis and Berry, 1987; Crofts and Horton, 1991). The magnitude of the trans-thylakoid
proton gradient depends on the ATP/ADP turnover rate, which in turn depends on the
activity of the Calvin -Benson cycle and on the alternative pathways of photosynthesis,
such as photorespiration, Mehler reaction, nitrogen- and sulphur-assimilation, and the
malate valve (Huppe and Turpin 1994; Edwards and Walker 1983; Scheibe and
Sonja VELJOVIû-JOVANOVIû: MEHLER AND APX REACTIONS
505
Beck,1994). When CO2 uptake is blocked under stomatal closure, the activity of the
Calvin-Benson cycle depends on the CO2 supply from respiratory and photorespiratory
metabolism. It has been shown that the contribution of the alternative pathways
(nitrogen- and sulphur-assimilation and malate valve) is minor when photorespiration
was inhibited (Loreto et al., 1994; Brestic et al., 1995; Valentini et al., 1995). Under
aerobic conditions the main electron acceptor of photosynthetically generated reducing
equivalents is oxygen: directly participating in the Mehler reaction (Mehler, 1951) or
competing with CO2 for RuBP at the active site of Rubisco (Jordan and Ogren, 1984).
A protective role of O2 against photo-inactivation has been shown (Ziem-Hanck and
Heber, 1980; Asada and Takahashi, 1987). However, there has been some dispute in
the literature whether the Mehler reaction or the photorespiration is more important in
protecting the photosynthetic apparatus against photoinhibition in situ (Wu et al., 1990;
Horman et al., 1994). A coupling of the Mehler reaction to the ascorbate peroxidase
reaction in thylakoids (reactions shown in Fig. 1), forming the water-water cycle
(Asada et al., 1998), accomplishes an efficient energy dissipation cycle, which prevents
over-reduction in the chloroplasts and creates a trans-thylakoid proton gradient in
isolated chloroplasts (Schreiber and Neubauer, 1990; Schreiber et al., 1991). It has
been shown in leaves that these process can account for the bulk of photon utilization
upon increasing light intensities (Canvin et al., 1980; Badger, 1985).
Therefore, oxygen has a dual role in photosynthesis:
it is a potentially dangerous molecule, as it can be photoreduced in the
&
thylakoid membrane to produce reactive oxygen species and cause oxidative
damage;
it is one of the main electron acceptors which is used in alternative biochemi&
cal pathways, preventing over-reduction and photo-inactivation of PSII.
GENERATION OF ACTIVE OXYGEN SPECIES
IN PHOTOSYNTHESIS
Photoreduction of dioxygen by the primary electron acceptor in the PSI complex
is the main source of O2.- in illuminated chloroplasts (Elstner and Heupel, 1975; Asada,
1994). The rate of photoproduction of O2.- in the Mehler reaction depends on the source
(thylakoids, chloroplasts or leaf), and on the presence of electron acceptors, and varies
from 4 to 20% of total electron transport (Furbank and Badger, 1983; Robinson, 1988;
Hodgson and Raison, 1991). Much higher rates of “the Mehler photorespiration”
(Osmond and Grace, 1995) were observed when gross O2 evolution was measured in
air by leaves (Canvin et al., 1980; Badger 1985).
A reaction of O2 with free radicals is also possible. In illuminated chloroplasts,
paraquat (1,1-dimethyl-4,4-bipyridylium) is photoreduced in PSI to its cation radical
(Em= -440 mV) which reduces O2 yielding O2.- (Farrington et al., 1973). Increased
generation of O2.- leads to increased production of H2 O2 which can diffuse to the
stroma inhibiting photosynthesis (Neuhaus and Stitt, 1989).
Formation of singlet oxygen in illuminated chloroplasts and its effects on photoinactivation was first observed by Takahama and Nishimura (1975). Singlet oxygen
was suggested to play an important role in damaging the D1 protein (Durrant et al.,
1990; Macpherson et al., 1993; Hideg et al., 1994).
In chloroplasts, H2O2 is mainly produced from O2 .- catalyzed by superoxide
dismutase (SOD) at a site where O2.- is photoreduced, within the thylakoid membrane
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IUGOSLAV.PHYSIOL.PHARMACOL.ACTA, Vol. 34, No.2, 503-522, 1998
(Asada and Badger, 1984). During photorespiration, the glycolate produced from
ribulose 1,5-bisphosphate is oxidized by the glycolate oxidase in peroxisomes.
Generated H2O2 is scavenged in peroxisomes by the catalase which catalyzes the
disproportionation of H2O2 to H2 O and O2 . Inhibition of CO2 fixation by H2 O2 (Kaiser,
1976) has been found to be due to the oxidation of thiol groups of several enzymes
(fructose 1,6-bisphosphatase, NADP-glyceraldehyde 3-phosphate dehydrogenase,
ribulose 5-phosphate kinase and sedoheptulose 1,7-bisphosphatase) by H2O2 (Kaiser,
1979; Charles and Halliwell 1981; Tanaka et al., 1982). The rate of H2O2 production
in the Mehler reaction is sufficiently high to cause an accumulation of 10 µM hydrogen
peroxide within 0.5 s (causing an inhibition of CO2 fixation up to 50%) in the case
when the scavenging enzymatic system of the chloroplasts does not function (Asada,
1994).
Oxidative stress might be also the result of enhanced production of .OH in the
Fenton reaction. If H2O2 is not properly scavenged, in the presence of increased
concentrations of transition metals such as Fe2+ or Cu+, there is a risk of generation of
highly reactive hydroxyl ions and non-selective oxidation of cellular constituents. In
chloroplasts, iron is stored in the form of phytoferritin which is unable to react with
H2O2 (Bienfait and van der Mark, 1983). But under certain stress conditions an excess
of Fe2+ and Cu+ has been detected (Price and Hendry, 1991; Moran et al., 1994).
WATER-WATER CYCLE
The photoreduction of dioxygen to water in PSI is coupled with the photooxidation
of water in PSII (Fig. 1).
The cycle consists of the following reactions (Asada et al., 1998):
(photooxidation of water in PSII)
2H2O O2 + 4[e-] + 4H+
(photoreduction of O2 in PSI)
2[e-] + 2O2 2O2.(SOD-catalyzed disproportionation of O2.- )
2O2.- + 2H+ H2 O2 + O2
(peroxidase-catalyzed reduction of H2O2)
H2O2 + 2AH2 2AH + 2H2 O
(regeneration of the electron donor for peroxidase)
2AH + 2[e-] + 2H+ 2AH2
----------------------------------------------------------(water-water cycle)
2H2O + O2 O2 + 2H2 O
SOD-catalyzed dispropotionation of O2.-, at a diffusion-controlled rate in the
vicinity of the producing site of O2.- in PSI , prevents the diffusion of O2.- to the stroma
(Ogawa et al., 1995).In plant tissues three types of SOD have been found with respect
to the prosthetic metals: CuZn-SOD, Mn-SOD and Fe-SOD. Mn-SOD is localized in
mitochondria and Fe-SOD in chloroplast stroma. As to CuZn-SOD, two isozymes were
found, cytosolic and chloroplastic (Kanematsu and Asada, 1990), the chloroplastic
CuZn-SOD being attached to the stroma side of thylakoid membrane. It has been
shown recently, using immunogold labeling (Ogawa, et al., 1996), that the "cytosolic"
isozymes are localized in the apoplast, in the nucleus and near the tonoplast.
The electron donor in the peroxidase reaction is ascorbate and regeneration of
ascorbate takes two alternative pathways: one directly from monodehydroascorbate and
the other via dehydroascorbate which is reduced by NADPH through the glutathioneascorbate cycle (Foyer and Halliwell, 1976; Groden and Beck, 1979).
Sonja VELJOVIû-JOVANOVIû: MEHLER AND APX REACTIONS
507
Fig. 1. Production of superoxide, hydrogen peroxide and monodehydroascorbate
during photosynthesis and systems for scavenging O2.-, H2 O2 and monodehydroascorbate (modified from Polle, 1995 and Asada et al., 1998). Numbers refer to the
following enzymes: 1 - CuZnSOD; 2 - tAPX, form of ascorbate peroxidase bound to
the thylakoid membrane; 3 - sAPX, soluble form of ascorbate peroxidase; 4 - MDAR,
monodehydroascorbate reductase; 5 - DHAR, dehydroascorbate reductase; 6 - GR,
glutathione reductase; Calvin-Benson cycle enzymes: 7 Fd NADPH reductase; 8 Rubisco, ribulose bisphosphate carboxylase oxygenase; 9 - GAPDH, glycer-aldehyde
phosphate dehydrogenase; 10 - PGK - phosphoglycerate kinase; 11 - RuPK -,
phosphoribulosekinase. Enzymes which are activated by the ferredoxin-thioredoxin
system and can be inactivated by H2O2 are GAPDH , FBP-ase (fructose bisphosphatase), SBP-ase (sedulosobisphosphatase) and RuPK. Xanthophyll cycle located within
thylakoid membrane: violaxanthin (V) is de-epoxidised to zeaxanthin (Z) by the
enzyme violaxanthin de-epoxidase (VDE). A part of the photorespiratory metabolism
(glycolate pathway) is located in peroxisomes (PX), where a substrate for oxidation
(glycolate) by glycolate oxidase (13) is transported from chloroplasts. One of products
is H2O2 which is scavenged by catalase (14) within peroxisomes. In mitochondria
(MIT) glycine is decarboxylated yielding CO2. Under stomatal closure the CO2
released during photorespiration is reassimilated in the Calvin-Benson cycle.
Monodehydroascorbate can be regenerated to ascorbate via several reactions:
(1) acid-catalyzed spontaneous
2MDA + H+ AsA + DHA
disproportionation
(2) (Miyake and Asada, 1994)
2MDA + 2 Fdred 2AsA + 2 Fdox
+
+
2MDA + NAD(P)H + H 2AsA + NAD(P) (3) by NAD(P)-dependent MDR
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IUGOSLAV.PHYSIOL.PHARMACOL.ACTA, Vol. 34, No.2, 503-522, 1998
Reactions 2 and 3 both consume reducing equivalents from the photosynthetic electron
transport chain. Thus, monodehydroascorbate and NADP+ compete for the photoreduced ferredoxin in PSI. This ferredoxin-dependent reduction of mono-dehydroascorbate, together with reactions catalyzed by CuZnSOD and ascorbate peroxidase
form a thylakoid scavenging complex (Asada et al., 1998) and provide a very efficient
sink for electrons from PSI. Monodehydroascorbate reductase preferably uses NAD to
NADP and is not found to be bound to the thylakoid membranes. This enzyme,
together with glutathione reductase and dehydroascorbate reductase, forms the stromal
scavenging system for H2O2 (Asada et al.,1998).
ROLE OF THE MEHLER-PEROXIDASE REACTION IN
DISSIPATION OF EXCESS LIGHT ENERGY
Photoreduction of O2 in aerobic organisms is an inevitable event, due to its high
affinity for the PSI reducing site (Km = 2 to 85 µM, Furbank et al., 1982). In earlier
literature much attention has been devoted to the role of oxygen photoreduction in ATP
synthesis by creating a trans-thylakoid proton gradient and in the poising of the cyclic
electron transport carriers stimulating their activity (Heber 1973; Heber et al., 1978;
Ziem-Hanck and Heber, 1980). A photo-protective role of the O2 uptake in high light
was proposed by Radmer and Kok (1976). However, the Mehler reaction alone cannot
mitigate the photoinhibition, as it saturates at low light and its rate is about 7% of the
total photosynthetic electron flux in isolated thylakoids (Asada and Takahashi, 1987).
Neubauer and Schreiber (1989) showed that in intact isolated chloroplasts
ascorbate peroxidase catalysed reduction of externally added H2O2 can be as efficient
as paraquat-catalysed O2-reduction. The importance of coupling of the Mehler reaction
to the ascorbate-peroxidase reaction for the acceleration of the ûpH generation and
down-regulation of PSII has been proposed by Schreiber and Neubauer (1990). The
Mehler-peroxidase reaction rate increased with increasing light intensity and it did not
saturate even at high light intensities (Hormann et al., 1994; Forti and Elli, 1996).
When compared to the Mehler reaction alone, the Mehler-peroxidase reaction
creates a greater pH gradient, as both the dismutation of O2.- and the regeneration of
ascorbate consume protons at the stromal side of the thylakoids (Asada et al., 1998).
Photoreduction of O2 can be greatly accelerated by MDA reductase (Miyake and
Asada, unpublished results cited in Asada, 1998). Neubauer and Yamamoto (1992)
demonstrated that the Mehler-peroxidase reaction driven pH gradient mediates the
formation of zeaxanthin.
In the water-water cycle, NADPH is consumed when ascorbate is regenerated
from DHA in the glutathione-ascorbate cycle, and from monodehydroascorbate in a
reaction catalyzed by the monodehydroascorbate reductase (Fig.1). Besides,
monodehydroascorbate itself appears to be a powerful Hill reagent (the photoreduction
rate constant of monodehydroascorbate is 40 times greater than that of NADP (Miyake
and Asada, 1994). Thus, reduction of H2O2 by ascorbate with ascorbate peroxidase and
subsequent regeneration of ascorbate provides an efficient mechanism for the dissipation of excess energy. It is assumed that the Mehler-peroxidase reaction is the main
alternative sink beside the photorespiratory pathway (Osmond and Grace, 1995).
Sonja VELJOVIû-JOVANOVIû: MEHLER AND APX REACTIONS
Fig. 2. A system for simultaneous measurement of gas exchange, chlorophyll
fluorescence and absorbance changes from an intact leaf during photosynthesis. A part
of a leaf was enclosed in a thermostated sandwich-type cuvette (T) which allows
controlled gas flow over one side of a leaf. The composition of the gas stream passing
over the leaf was adjusted by mass-flow controllers (1-6). Transpiration and CO2
exchange were recorded by an infra-red analyzer (IRGA) in the differential mode. The
leaf was illuminated from a halogen lamp (L3) by means of fiber optics which
simultaneously transmitted optical signals from the leaf to receiving devices.
Modulated chlorophyll fluorescence was measured by a Walz GmbH fluorimeter (PAM
1, 2). To obtain an estimate of the extent of different kinds of fluorescence quenching
(photochemical: qQ, and nonphotochemical: NPQ) the saturation pulse method, using
L1-L2 lamps, was employed (Bradbury and Baker, 1981). Absorption of nonmodulated weak green measuring beam was measured by a photomultiplier (PM)
equipped with filters to obtain green light peaked at 545 nm for recording light
scattering or 505 nm for recording changes in zeaxanthin level.
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IUGOSLAV.PHYSIOL.PHARMACOL.ACTA, Vol. 34, No.2, 503-522, 1998
THE ACTIVITY OF ALTERNATIVE ELECTRON FLOWS
IN A LEAF
Determination of the activity of Mehler-peroxidase in situ by measuring gas
exchange is not possible, since O2 uptake occurs simultaneously with O2 evolution. It
was only by using mass spectrometry and different isotopes of oxygen which enabled
measurement of the Mehler-peroxidase reaction directly (Canvin et al., 1980). Another
indicator of the Mehler-peroxidase activity can be the detection of monodehydroascorbate signal from intact leaf by EPR.
Indirectly, it is possible to determine the activity of alternative electron flows by
measuring photochemical activity of PSII and CO2 assimilation rates simultaneously,
and then to compare the estimated total electron flow to that going to the CalvinBenson cycle (Krall and Edwards, 1992; Kingston- Smith et al., 1997). It is important
to measure gas exchange and chlorophyll fluorescence simultaneously from the same
part of leaf. Figure 2 shows a scheme of apparatus designed for such nondestructive
measurements of photosynthesis.
Leaves of spinach (Spinacea oleracea L.) were treated with aminotriazole or
fumigated with SO2 and photosynthesis and chlorophyll fluorescence measurements
were performed in air and at low oxygen (1%). Inhibition of photosynthesis by both
aminotriazole and SO2, had different characteristics in photorespiratory, when
compared to non-photorespiratory conditions. Analysis of chlorophyll fluorescence
quenching with varying irradiance at 21% O2, showed only a small decrease in PSII
efficiency when inhibition of CO2 uptake by aminotriazole was considerable. At low
oxygen, when photorespiration was prevented, neither CO2 uptake nor PSII activity was
affected by aminotriazole. The observed decline in CO2 uptake only in photorespiratory
conditions could be explained by the toxic effect of H2O2 on photo-synthesis which
leaked out from peroxisomes to chloroplasts when catalase was inhibited by
aminotriazole. The calculated electron flux through PSII in aminotriazole treated leaf
at 21% O2 indicated that a large proportion of electrons was directed to nonassimilatory electron transport (Fig. 3).
Short-termed fumigation of a leaf with SO2 caused a transient decrease in
photosynthetic CO2 uptake to a similar extent in air and at low oxygen (Fig. 4).
However, when data for CO2 assimilation rate during inhibition and recovery processes
were plotted against PSII efficiency, an increase in non-assimilatory electron transport
at 21% O2 became evident in the change in slope of the relationship between the rate
of total electron flow and flux of electrons through the carbon pathway (Fig. 4).
Analysis of chlorophyll fluorescence quenching with varying irradiance demonstrated
a difference between the calculated electron flux through PSII and electron flux directed
to carbon reduction in SO2 fumigated leaves depending on the activity of photorespiration. At 21% O2 the alternative electron sink was indicated in presence of SO2.
This difference, observed in leaves treated with SO2 or aminotriazole (Fig 3, 4), and
due to non-assimilatory electron flow, can be explained by the occurrence of the
Mehler-peroxidase reaction.
Sonja VELJOVIû-JOVANOVIû: MEHLER AND APX REACTIONS
511
A
18
100
10
Jf/A
Jf (P mol m-2s-1)
14
80
60
40
6
20
0
0
2
10
20
30
40
50
60
70
0
Jc (min) (P mol m-2 s-1)
200
400
200
400
600
P mol photons m-2 s-1
800
B
100
16
Jf ( Pmol m-2 s-1)
80
12
Jf/A
60
8
40
4
20
0
0
10
20
30
40
50
Jc(min) (P molm-2s-1)
60
0
0
600
P mol photons m-2 s-1
800
Fig. 3. The effect of aminotriazole on the relationship between the rate of total electron flow, Jf
and the flux of electrons through the carbon pathway, Jc (min) estimated from chlorophyll
fluorescence and CO2 assimilation rate, respectively under varied light intensities in 21% O2 (A)
and in 1% O2 (B). Open symbols denote control leaves and closed denote leaves treated with
2 mol·m-3 aminotriazole.
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IUGOSLAV.PHYSIOL.PHARMACOL.ACTA, Vol. 34, No.2, 503-522, 1998
A
-2 -1
Jf ( P mol m s )
150
10
8
90
6
Jf/A
120
60
4
30
2
0
0
10
20 30 40 50 60
Jc(min) ( P mol m-2 s-1)
0
0
70
200
400
P mol m-2 s-1
600
800
B
120
10
Jf ( Pmol m-2 s-1)
100
8
80
Jf/A
60
6
40
4
20
2
0
0
10
20
30
40
50 60
Jc(min) (Pmol m s )
-2 -1
70
0
0
200
400
P mol m-2 s-1
600
800
Fig. 4. The effect of SO2 on the relationship between the rate of total electron flow, J f and flux
of electrons through the carbon pathway, Jc (min) estimated from chlorophyll fluorescence and
CO2 assimilation rate, respectively, under varied light intensities in 21% O2 (A) and in 1% O2
( B). Open symbols denote values from control leaves and closed symbols denote values from
leaves fumigated with 4 µ l-1 SO2.
MONODEHYDROASCORBATE:
AN INDICATOR OF OXIDATIVE STRESS IN A LEAF
In illuminated chloroplasts the major reaction producing monodehydroascorbate
is an ascorbate peroxidase-catalyzed univalent oxidation of ascorbate (Hossain et al.,
1984). There are several additional reactions in chloroplasts in which monodehydro-
Sonja VELJOVIû-JOVANOVIû: MEHLER AND APX REACTIONS
513
ascorbate can be generated. In the reaction of deepoxidation of violaxanthin to
zeaxanthin (Fig. 1) ascorbate is a cofactor providing reducing equivalents. It has been
shown that ascorbate peroxidase and violaxanthin depoxidase compete for ascorbate
(Neubauer and Yamamoto, 1994) and that monodehydroascorbate is formed during the
reaction (Miyake and Asada, 1988). It has also been shown that ascorbate can be an
endogenous electron donor to PSII when the donor side is inactivated and that in this
reaction it is photooxidized to monodehydroascorbate (Mano et al., 1997). Mano
(1998) has also suggested that ascorbate can be an electron donor for oxidized P700
when down-regulation of electron transport is in concert. A high reactivity of ascorbate
.
with radicals (.OH, GS-., organic radicals: RC. , ROO. , RO. , lipid radicals: L,
LOO. , LO. )
makes possible those reactions in chloroplasts yielding monodehydroascorbate (Bielski,
Figure 5. EPR spectra of Vicia faba. leaves treated with: A. paraquat, B. amino-triazole, and
C. sulfite. The irradiance was 1000 W·m-2. Numbers in A. and B. mark the time in light when
EPR spectra were recorded.. In C: trace 1 - control leaf in dark; trace 2 - control leaf in the
light; trace 3 - sulfite treated leaf in dark; trace 4 - sulfite treated leaf in the light. EPR spectra
in C-2 and -4 were recorded after 40 s in the light. Data extracted from Veljoviü-Jovanoviü et
al., 1998.
1982). Regeneration of tocopherol during scavenging of lipid radicals by ascorbate
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could also lead to mono-dehydroascorbate generation in the thylakoid membrane
(Asada et al.,1998).
As monodehydroascorbate is a long lasting anion radical, it can be detected by
EPR spectroscopy at room temperature and serve as an endogenous probe for oxidative
stress (Buettuer and Jurkiewicz, 1993). Under optimal conditions, the concentration of
monodehydroascorbate is such that it could not be detected in the leaf in the light or
darkness (Heber et al.,1996). Thus, when the EPR cavity, enabling diffusion of CO2
through a part of an enclosed leaf , was illuminated with light of very high intensity
(8000 µmol E·m-2 ·s-1), monodehydroascorbate was not detected (Fig 5). The
accumulation of monodehydroascorbate in leaves could only be induced by various
factors which cause oxidative stress (Westphal et al., 1992; Stegmann et al., 1993;
Heber et al., 1996; Hideg et al., 1997; Veljoviü-Jovanoviü et al., 1998). Under
environmental stress when the rate of production of oxygen activated species surpasses
the monodehydroascrobate reducing capacity, monodehydroascorbate can be detected
by EPR, as was the case in leaves treated with paraquat and aminotriazole (Fig. 5A and
B). In illuminated leaves paraquat is photoreduced to the paraquat radical that rapidly
reacts with O2 to give O2.- (Farrington et al., 1973). Superoxide is disprotonated to H2 O2
by SOD at the site of its production, leading to an increase of H2O2 level in the chloroplasts. Scavenging of H2O2 gives rise to monodehydroascorbate signal that is light
dependent (Fig. 5).
SCAVENGING OF H2O2 FROM PEROXISOMES
IN CHLOROPLASTS
The concept of delocalized scavenging of H2O2 has been demonstrated in
experiments with paraquat-treated leaves, when H2O2 formed in chloroplasts was
scavenged in vacuoles (Takahama and Egashira, 1991). Another source of H2O2 in
illuminated leaves is the reaction of oxidation of glycolate to glyoxalate (Fig.1). By
inactivation of catalase it could be possible to test whether H2O2 generated in peroxisomes is reduced by ascorbate in chloroplasts using reducing equivalents from the
photosynthetic electron transport chain for the regeneration of monodehydroascorbate
or dehydroascorbate. A stimulated non-assimilatory electron flow, occurring parallel
with the inhibition of CO2 assimilation (as shown in Fig. 3 and 4), could be explained
by such a mechanism.
A novel isozyme of ascorbate peroxidase has been found on the peroxisomal
membranes in pumpkin (Yamaguchi et al., 1995). When catalase is inhibited by sulfite
(Veljoviü-Jovanoviü et al., 1998; Milovanoviü, 1996) or by aminotriazole (Allen and
Whatley, 1978), the H2O2 generated in peroxisomes could leak through the pores in
peroxisomal membranes (Reumann et al., 1998) out of peroxisomes and be reduced by
ascorbate peroxidase in the cytosol or on the peroxisomal membrane. Therefore, the
monodehydroascorbate signal, detected in sulfite- and aminotriazole-treated leaves, can
originate from these reactions. However, monodehydroascorbate could also be
generated in the reaction of reduction of H2O2 by ascorbate in chloroplasts (Fig. 1). In
illuminated leaves peroxisomes are tightly appressed to the chloroplasts enabling H2O2
to also diffuse to the stroma of chloroplasts, where it exhibits its inhibitory effect on
the Calvin-Benson cycle enzymes and intermediates (Fig. 3, 4), but might be also
scavenged by ascorbate peroxidase generating monodehydroascorbate (Fig. 5).
The inhibition of photosynthesis at air level oxygen concentrations, when
Sonja VELJOVIû-JOVANOVIû: MEHLER AND APX REACTIONS
515
compared to 1% O2, is also larger and faster when leaves were fumigated with SO 2
(Veljoviü-Jovanoviü, 1995). The observed transient stimulation of photosynthesis
inhibition in 21% O2 can be explained by the additional effect of H2 O2 on the CalvinBenson cycle (Kaiser, 1976). A profile of metabolites measured in leaf extracts and in
chloroplasts isolated from leaves after fumigation with SO2 (Veljoviü-Jovanoviü et al.,
1993) is similar to that obtained in chloroplasts after addition of H2O2 (Heldt et al.,
1978; Kaiser, 1979), or in intact leaves treated with paraquat (Neuhaus and Stitt, 1989).
A slight increase in the alternative electron flow (shown in Fig. 3, 4) might be an
indicator of H2O2 scavenging in chloroplasts of intact leaves.
A stimulation of zeaxanthin synthesis by the Mehler-peroxidase reaction activity
has been shown by Neubauer and Yamamoto (1992). Thus, additional evidence for the
stimulation of the ascorbate peroxidase reaction in leaves fumigated with SO2 can be
sought in the observed transient increase in zeaxanthin synthesis which occurs only in
photorespiratory conditions (authors unpublished results).
Although the amount of catalase in peroxisomes is quite large, it has been shown
that it decreased its activity in some stress conditions (Omran, 1980; MacRae and
Ferguson, 1985; Feirabend et al., 1992; Schoner and Krause, 1990). It also has been
shown that the turnover of catalase is similar to that of D1 protein which is the
indicator of photoinhibition (Feirabend and Dehne, 1996).
Consequently, utilization of reducing power of chloroplasts, in potentially photoinhibitory conditions, to reduce H2O2 formed in peroxisomes, might be considered as
an additional mechanism of dissipation of excess light energy.
CONCLUSIONS
Decrease in CO2 assimilation in leaves under various stress conditions is usually
accompanied by a concomitant decrease in PSII activity, an increase of thermal
dissipation processes, and an increase of the activity of alternative photosynthetic
pathways other than CO2 assimilation. Two pathways, draining the surplus of electrons,
are the main candidates for the prevention of over-reduction:
the Mehler-peroxidase reactions;
&
the photorespiratory pathway.
&
Difficulties are encountered when attempting to measure and differentiate between
the activity of these two processes. Uptake of oxygen can be resolved from oxygen
evolution by use the of mass spectrometry and oxygen isotopes. Such an approach
demonstrated increased rates of O2 consuming reactions under stress conditions.
However, resolving of individual contributions of different O2 consuming reactions
(chloral respiration, mitochondrial respiration, photorespiratory O2 uptake) in the light
is not yet possible. Simultaneous use of sophisticated techniques such as photosynthetic
fluorescence analysis, infra-red gas analysis and EPR spectroscopy could help in
determining the contribution of the Mehler-peroxidase reactions to the decrease of
excess light energy in the leaf.
The mechanism of catalase inhibition which occurs in many oxidative stresses and
photoinhibitory conditions, and its possible interaction with the processes in
chloroplasts, remains to be resolved. Inhibition of catalase should lead to increased
levels of cellular H2O2, this being an important signal molecule involved in regulation
of transcription of enzymes involved in antioxidative metabolism.
Acknowledgments. - This work was supported by the research grant 03E221 from
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IUGOSLAV.PHYSIOL.PHARMACOL.ACTA, Vol. 34, No.2, 503-522, 1998
the Ministry of Science and Technology of Serbia. The author is grateful to Dr.M.
Plesniþar, Dr.D.Pankoviü and Dr.ä.Vuþiniü for helpful comments.
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Received November 13, 1998
Accepted November 23, 1998