j. Plant Physiol. Vol. 148. pp.

249-257(1996)
Mechanisms of Oxygen Activation during Plant Stress:
Biochemical Effects of Air Pollutants
SUSANNE HIPPELI and ERICH F. ELSTNER
Lehrstuhl fur Phytopathologie, Technische Universitat Munchen, 85350 Weihenstephan, Germany
Received June 6, 1995 . Accepted September 15, 1995
Summary
Green plants can adapt to drought, temperature, high and low light, infections, air pollurion and soil
contamination. Dependent on the strength of these impact(s), several symptoms indicate the deviation
from normal, steady- state-metabolism. Most of these visible or measurable symptoms are connected with
oxygen activation where principally a transition from heterolytic (two electron transitions) to increased
homolytic (one electron transitions) reactions is observed. Homolytic reactions create free radicals, which
are generally counteracted by a parallel increase of radical scavenging processes or compounds thus war
ranting metabolic control within certain limits. At advanced states of stress control may be gradually lost
and chaotic radical processes dominate. Finally, cellular decompartmentalizations allow lytic and necrotic
processes. Every episode during this cascade is characterizable by the balance between pro- and antioxid
ative capacities. Photosynthetic processes which are under metabolic and oxygen-detoxifYing control are
converted into photodynamic reactions which are only controlled by light and scavenger- and/or
quencher-availabilities. Photoinhibition may represent the threshold between these two light driven
events. This (more or less theoretical) sequence of events is not yet fully understood and in most cases can
only punctually be characterized and followed by indicator reactions such as ESR.
In this communication we shall concentrate on basic redox-mechanisms during oxidative stress situa
tions involving primary air pollutants. Chemical interactions between different gaseous pollutants and
generally mechanisms of the finally toxic effects on target molecules will be shown.
Key words: Airpollution, photooxidations, plant stress.
Abbreviations: DCMU =Dichlorophenyl-dimethylurea; DOPA =3,4-Dihydroxyphenylalanine; ESR =
electron spin resonance; PA =primary receptor; PAN =peroxyacetyl nitrate; SH =sulphhydryl; VOC =
volatile organic compounds.
1. Induction of plant stress
The term stress stems from physics and has been uncriti
cally introduced in medicine and botany. In general mechan
ics it is clearly defined as the point or degree of bending of an
elastic system just between symptomless reversibility and irre
versible deformation or break. In medicine and botany, stress
is supposed to indicate all situations beyond normal, defined
by the observer i.e. man.
All organs of higher plants (with some exceptions) perform
aerobic metabolism and are thus subject to activated oxygen
species, oxygen-oversaturation" and thus possibly oxygen
stress. This may occur under high light intensities over
longer time periods, during drought periods, after poisoning
such as soil contamination or air pollution or under mineral
deficiency. A well-balanced antioxidative strategy has been
elaborated during some 3 to 4 billion years. In this review
basic reactions operating during oxidative stress situations
will be discussed where certain prooxidative situations in
plant life, air pollution effects and antioxidative processes will
be dealt with.
Plant stress underlies sincerely a different definition as
compared to what humans may define for themselves. It is
difficult to exactly define the term stress, however, since it is
1996 by Gustav Fischer Verlag, Srurtgarr
250 SUSANNE HIPPELi and ERICH F. ELSTNER
Biological influence
UV high intensIty flowering
low intensity fruit ripening
I. 7:. II
insects
infections
allelopathy factors
competition
I Magnetic Fields? Moon Sun I '

Hydratioii] Chemical Factors I Mechanical factors
'-----'---'
high drought salts wind
flooding heavy metals lightning
./ freezing
pH fire
10.", Fig. 1: The seven stressfields for plants
chilling air pollutants snow
cutting. biting
(from Eistner et al., 1988; Elstner and Oss
pressing
wald, 1994).
Impact. "Stress",
Plant

Hormonal Responses:
Synthesis or Degradation

I
abscisic acid yt k' .. ethylene
c t Inln /
Changes in Metabolism' /
stomatal opening thickening of axes
root permeability abscision
prolin content pigment bleaching
sugar content protein turnover
secondary metabolites lipid turnover
___ lignification
----"'1 Plant Development ,
Fig. 2: Stress induced signal transduction
increase in stability. decrease in waterloss. increase in water uptake.
increases in tolerance and avoidance reactions.
and plant development (from Eistner et al.,
allelopathic reactions (emissions) 1988; Eistner and Osswald, 1994).
used for the description of processes and syptoms during and preconditions, developmental processes, stress and oxy
which the individuum is supposed to be in a more or less gen activation are tightly connected with each other in sev
critical situation or to suffer, undoubtedly coupled with mes eral, up to date only partially understood ways. Wounding or
sage feedback between different organs. The individual plat other mechanical influences such as bending, increased trans
forms indicating the start depends on the genetic outfits and port system resistance induce oxidative processes such as fatty
the amount of impact necessary. It thus is almost impossible acid peroxidation through partial or complete decompart
to exactly define fixed stress points i.e. the thresholds between mentalization and induction or inactivation of detoxification
normal metabolism and stress metabolism. In the following pathways. Similarly, ionizing and UV radiation, drought,
an attempt is made to more closely look at the most charac flooding, osmotic impacts (high salt concentrations, desicca
teristic metabolic features underlying principle routes and tion), deficiency in macro- or micro nutrients, dramatic tem
reactions operating close to the above mentioned threshold. perature changes such as heating, chilling or freezing as well
Seven stressfields have been defined for plants (Fig.I). as poisoning (air pollution, water and soil pollution, herbi
cide treatment) cause changes in pro- and antioxidative
potentials. These are responded by hormone synthesis or
2. Fundamental metabolic conditions governing release from endogenous stores inducing increased resistance,
stress situations stability, avoidance, tolerance or repair (Fig. 2).
All the above mentioned influences on plants underly feed
As outlined and reviewed by several authors and editors back to the chloroplast: independent on the site of transport
(Elstner et al., 1988; Pell and Steffen, 1991) ecological factors or metabolic block, be it in the roots (salt, drought, mineral
Oxygen Activation during Plant Stress 251
Saccharose
GAP
"- ADP PI ATP
Fig. 3: Basic feedback events finally block
ing photosynthetic electron transport (from
Pi
Elsrner et al., 1988; Elsrner and Osswald,
1994).
deficiency), in the transport system (xylem or phloem blocks
by infections) in phloem loading (photooxidants, infections)
limitations in CO
2
fixation (stomatal closure, lack of Calvin
cycle activities due to enzyme inhibition) or in photosyn
thetic electron transport, photosynthetic oxygen activation
and/or photodynamic processes are provoked, leading to a
chain of reactions outlined in a following section. A summary
of the mentioned feedback system is given in Fig. 3.
The situations indiCated in Fig. 3 has been simulated with
isolated chloroplasts in the presence or absence of the natural
electron acceptor, ferredoxin + NADP and the electron trans
port blocker, DCMU. Bleaching of photosynthetic pigments
and formation of ethane from peroxidized linolenic acid
(Konze and Elsrner, 1978; Elsrner and Pils, 1979, Elsrner and
OSwald, 1980) are effects of electron transport limitations.
Several lines of evidence lead us to conclude (Elsrner et aI.,
1988) that electron flow through photosystem II under pro
duction of hydrogen peroxide is not initiating pigment
bleaching but electron block after photosystem II initiates
bleaching via lipid peroxidation by singlet oxygen. This also
lead us to assume that block of the electron transport is the
final stressing condition for green plants. Before this occurs,
a series of metabolic and regulatory activities are passed. Mas
sive production of singlet oxygen in the photosystems is thus
a function of impaired metabolism allowing a definition of
plant stress and metabolic events at the threshold between
fully reversible (controlled) and only partially reversible
(chaotic) reaction sequences from a radical view.
From the recent literature it is evident that continuing
stress leads to changes in metabolism (see above), regulation
and morphological symptoms, indicating changes in the lim
its of anabolic, catabolic, amphibolic and anaplerotic path
ways. In the course of stress development a maximum of
steering activities is achieved, concerning the establishment
of tolerance, avoidance, and detoxification by opening the
stress- and rescue boxes. In the following, oxidative processes
yield an aging of the most strongly stressed compartments
where parts of the regulatory functions are lost. At the end of
this line, aldehydes and lipofuscins are chemically produced
from peroxidized lipids and protein amino groups. These
products contribute to cell necrotization in addition to phe
noloxidase and peroxidase products (i.e. o-quinones from
o-dihydroxy phenylpropanes such as DOPA and caffeic acid)
responsible for the browning. A summary of these events is
outlined in Fig. 4.
Oxygen is also activated under <<normal, metabolic steady
state conditions but especially in plant disease development
(Tzeng and DeVay, 1993). Thus, reactive oxygen species have
come into the focus of investigations on pathological and
toxicological situations both in human medicine and phyto
medicine since approximately two decades. (To mention just
a few: Michelson et aI., 1977; Bannister and Bannister, 1980;
Ciba Foundation Symposium, 1979; Bannister and Hill,
1980; Cohen and Greenwald, 1983; Greenwald and Cohen,
1983; Elstner, 1982, 1987, 1990, 1991, 1991 a; Gilbert, 1981;
Halliwell and Gurreridge, 1989; Hayaishi, 1974; Pell and
Steffen, 1991; Pryor, 1975-1982; ScandaIios, 1992; Sies,
1985, 1991).
3. Air pollutants as inducers of oxidative stress
The apparent local and global increases of certain atmos
pheric trace gases have become a world wide focus of discus
sion since all living organisms and ecosystems are concerned
by these atmospheric and stratospheric global changes
(Manning and v. Tiedemann, 1995). The role of oxidative
processes in air pollution has already been reported in the last
century where the production of acid rain was recognized as
an oxidation by atmospheric hydrogen peroxide of sulfur dio
xide stemming from coal combustion (Holle, 1892). Sim
ilarly, chlorophyll bleaching was described as a process cata
lyzed by oxidizing enzymes (Woods, 1899). Nevertheless we
were greatly surprised in our days by the overwhelming num
ber of recent findings that oxygen toxicity is the dominating
factor in general toxicology and pathology (Elsrner, 1990).
252 SUSANNE HIPPELI and ERICH F. ELSTNER
I META80USM 1 IREGULAnON I
noc fUly standard wIcllh
Ulllzed. 01 reglAallon
reglAar
foodback
anabolic +B->cataboIic
amphibolic
'socondary
routes'. new
prodoos
fUly
re
ver
si e
d etoxlflCallon
Clmetabolism
!
ethylene
Induced repa" anaplerolic
fUly (PAL POD. P450)
rev\!( Slr8S.S prOleil\$,
Sible $lomatal responses.
respiration Increased
SITUAnON BEYOND 'STRESS POINr7
o 0
$lomatal

hlrdered.
aIIemaltve resp!ratlon.
par ethylene burst. MACC
l\a1ly fttJC)(escence Increase
rever. PSU-degradallon.
sible 'plastoglowr.
anlhocyans. phy1oa1exines
1ipo1usclnS'
membrane de
gradation
ethylene, ethanol
$lrano ESRslgnaJs
l
ine. lonopla$l de$l rvctIon
vers ble cell melanin formation
"wound'
l1AA1!l
reaCllohs'
chemlcaJ condensations

4. General Biochemical Aspect of Reactions with Air

Pollutants
Basic chemical reactions of the most prominent potentially
toxic trace gases will be discussed. Biologically it is almost
impossible to differentiate between the individual influences
under natural conditions since there is no single influence of
just one component of air pollutants on aerobic cells. Never
theless we will separately discuss the influences of 502, NO
x
ozone and peroxyacetyl nitrate (PAN) on important target
molecules.
IMORPHOLOGY I
firSI symptoms.
epinasty. colour.
t:J. orOWlh
clear
disease
symptoms:
bleaching

browning
abscision
Fig,4: Metabolic, regulatory and morpho
secondary
logical changes during stress development
Infec110ns (from Elstner et aI., 1988; Elstner and Oss
wald, 1994).
necrOClc;
4.1 Dark Reactions
4.1.1 50
2
and Derivatives
50
2
is produced during combustion of organic materials
especially of coal. 50
2
and HS0
3
- are involved in radical
chain processes. HS0
3
- is a reductant which, however, can
accelerate oxidative processes by reducing peroxidic bonds,
thus producing anions and radicals. Principally, 50
2
or
HS0
3
- preferentially reacts with the following types of mole
cules:
Oxygen Activation during Plant Stress 253
(a) aldehydes and ketones, where hydroxysulfonates are
formed, which in turn are inhibitors of several enzymes;
(b) olefines, where sulfonic acids are formed and the double
bond is lost;
(c) pyrimidines under formation of dihydrosulfonates,
which may have mutagenic effects;
(d) disulfides under formation of S-sulfonates, where the
S-S-bridge is split.
(e) superoxide (02'-) under formation of the very reactive
disulfite radical:
In this reaction, superoxide may be substituted by transi
tion metal ions:
M
n
+ + -t M(n-1l+ + S03"
HS0
3
' is a powerful initiator of several radical chain pro
cesses such as lipid peroxidation. Under aerobic conditions,
the sulfate radical is generated from the sulfite radical:
S03'- + O
2
-t S05'
S05'- + -t S04'- + sol
S04'- +S03'- -t +S03'
Both radical anions may be involved in destructive reac
tions initiated by 50
2
expositions.
Most of the physiological effects can be induced by hydro
gen abstraction as the primary reaction. None of the above
mentioned reactions, however, seems to be solely responsible
for the observed toxic effects or symptoms. Of special impor
tance is certainly the reaction with preformed hydroperoxides
(see below).
50
2
is a highly water soluble gas which is rapidly hydrated
forming sulfite (Peiser and Yang, 1978):
HOW W
S02 (g)" S02 (aq) pK 176) HS0:3 pK
Sulfite modifies the cellular energy metabolism in mam
malian tissues by decreasing the cellular ATP pool; this may
be due to changing the function of pyridine and flavin
nucleotides (McManus et al., 1989).
If sulfite is not metabolized via sulfite oxidase, it may be
oxidized via radical chain mechanisms. In the presence of
oxygen, a series of SOx- and oxygen radicals are generated
(see below). This reactions induce lipidperoxidation and
numerous connected reactions.
In clean air, 50
2
is present in concentrations lower than
20 Ilg/m3. In heavily polluted areas close to coal power plants
concentrations up to 1200 Ilg/m3 may be observed. Conver
sion factors for 502: 1 ppm = 2670 Ilg/m3; ppb = Ilg/m3 X
0.375; Ilg/m3 = ppb X2.67.
4.1.2 NO
x
NO and N0
2
are free radicals. Due to their unpaired elec
trons several one electron reactions can be initiated where the
attack of olefinic structures (double bonds) is one characteris
tic. The first reaction is most probably the abstraction of a
hydrogen atom.
N0
2
+ -CH=CH-CH;r- -t HN0
2
+ -CH-CH-CH
1(02)
chain reaction
Addition reactions can also be observed:
I I
N0
2
+ -c=c- -t N0
2
-C-C-'
I I
1 I I I
N0
2
-C-C-' + 02-t N0
2
-C-C-00'
I I 111
chain reaction
The primary product of combustion is NO. Since NO and
N0
2
are tightly connected with each other in atmospheric
chemistry via a very complicated chain of reactions (see Hip
peli and Elsrner, 1992) involving ozone and a vast amount of
volatile organic molecules (VOC) calculating with activities
or concentrations of only NO or N0
2
is not very meaning
ful, however.
Conversion factors for N0
2
: I ppm = 1910 Ilg/m3; ppb =
IlgxO.523; Ilg/m3 = ppbx 1.91.
4.1.3 Ozone andperoxides (PAN)
Ozone is formed from oxygen and NO
x
catalyzed by VOC
in a chain of atmospheric events. Ozone (( - )0-0(+) = 0)
with a standard redox potential of about 2 volts is a strong
oxidant and a very reactive molecule due to its ionic reso
nance structure. Characteristic chemical reactions with bio
chemical relevance are the splitting of olefinic bonds and the
reactions with thiols. Ozonisation of olefines yields inter
mediary ozonides, which in turn decompose into ketones and
other products probably via the so-called Criegee zwitter
ions. The resonance structures allow additions of carboxylic
acids, alcohols and water, finally yielding different peroxidic
products:
,,/ "P-0-9/
c=c + 03-t C--C--t -C + C
/" /1 I" I I
ozonide t
" +
C=O-O
/
additions to the Criegee zwitterion
O-OH
I
O-OH + ROH -t -C-OR
"-t (+)
I
/
o
I II
+ RCOOH -t -C-0-C-R
1
O-OH
-t
"
/
c=o + H
2
0
2
peroxidic products
254 SUSANNE HIPPELI and ERICH F. ELSTNER
Thiol oxidation leads to
sulfonic acids or disulfides:
HOOC-CH-CHr-SH --24 CHr-S-S-CHz
. I I I
NH
z
HzN-C-H
I
H-C-NH
z
I
cysteine
1o} cystin
COOH COOH
HOOC-CH-CHr-SO}H
I
NH
z
cysteine sulphonic acid
Similarly, glutathione and proteinic SH-groups are oxi
dized thus drastically changing the antioxidative power and
cellular redox balances together with enzymic functions. The
oxidation of SH-groups is responsible for inhibition of the
lipid synthesis in mitochondria and microsomes: Acyl-CoA
thioesterases, -thiokinases and acyltransferases are blocked,
detectable as inhibition of the glycerol-3-phosphate acylation
(Peters and Mudd, 1982). Glycerinaldehyd-3-phosphate
dehydrogenase (GAPDH), a key enzyme for glycolysis, is also
inactivated, where the oxidation of SH-groups in the active
center is responsible for the rapid loss of function. In addi
tion, peripheric SH-groups, tryptophan, histidine and methi
onine groups are oxidatively damaged (Knight and Mudd,
1984) thus provoking proteolytic degradation. Ozone inacti
vates aI-proteinase inhibitor via methionine oxidation (Moh
senin and Gee, 1989), and lysozyme via the conversion of
tryptophan into N-formyl kynurenine (Dooley and Mudd,
1982). Due to its ionic resonance structure ozone is more
water soluble than oxygen. Therefore hydrophilic compart
ments may be the main site of its reaction. Under acidic con
ditions ozone is quite stable, under basic conditions it
decomposes into molecular oxygen. At 22C hundred ppm
ozone on a water surface yield micromolar solutions. One
may calculate that up to 10-
6
moles of ozone are dissolved in
55 moles HzO. In the presence of aromatic compounds 0
3
is
decomposed into OH radicaltype oxidants. Dependent on
the concentrations of NO, aromatic compounds and high
light intensities, 0
3
can increase strongly where up to 400
5001lg/m3 can be reached on hot and sunny days. Average
concentrations in rural areas during sommer days may be
between 20-80llg per m
3
, depending on daytime and alti
tude. In sommer 1994 in Germany episodes with 150
250llg/m3 0
3
have frequently been observed.
Conversion factors for 03: 1ppm = 2000Ilg/m3, ppb =
Ilg/m3xO.50l, Ilg/m3 = ppbX2,00.
Different peroxides are present in polluted air; in this
respect peroxyacetyl nitrate (PAN) and HzO
z
are of major
importance. In centers of photosmog air pollution such as
the Los Angeles region PAN may yield up to 5-20 % of the
corresponding ozone concentrations. Besides PAN other per
oxides such as peroxypropionyl-, peroxybutyryl- and also per
oxybenzyl nitrate have to be considered as plant-toxic species
in polluted air. PAN at physiological pH (7.2) has a half live
of 4.4 min. It decays base-catalyzed essentially yielding acetate
and nitrite:
PAN may react with the following chemicals and bioche
micals:
(a) thiol groups, especially in enzymes, such as the SH
groups of glucose-6-phosphate dehydrogenase; the sub
strates, Glu-6-P and NAD may partially prevent SH-oxi
dation. Smaller S-containing molecules are also oxidized:
methionine is converted into methionine sulfoxide and
liponic acid and coenzyme A are converted into the cor
responding disulfides. With glutathione one mole PAN
converts 3 moles ofSH-groups.
(b) PAN reacts with NADPH, where the oxidized com
pounds (NAD, NADP) do not seem to further react with
PAN.
(c) PAN reacts with olefines under formation of epoxides
and further products of lipidperoxidation:
PAN + olefine CH
3
COO' + NO
z
+ epoxide(s)
CH3COO' COz + CH3'
CH
3
'+LH L+CH
4
(d) Reactions with amines result in their acetylations:
o
II
PAN +RNH
z
Oz + HNO
z
+CH}-C-NH-R
Conversion factors for PAN: 1 ppm PAN = 4370Ilg/m3;
ppb =Ilg/m3xO.223; Ilg/m3 =ppbx4.37.
4.2 Light-driven Reactions
Like humans and animals, plants under several conditions
may suffer from stress, although these conditions by defini
tion may be completely different from animal systems (Elst
ner and Osswald, 1994). Besides drought, flooding, frost,
heat, soil chemicals and many others, air pollution represents
one major stressor. As not outlined in detail at this point,
most of the mentioned conditions under continuing light
influence yield an overreduction of the photosynthetic elec
tron transport chain due to limited NADPH
z
reoxidation.
Under conditions with limited NADP+ in the stroma of
the chloroplasts (i.e. strong light, transport limitations, ATP
uncoupling, inhibition of Calvin-cycle activities and others)
reductive and photodynamic oxygen activation are measur
able. Especially inhibited COyfixation due to stomatal clo
sure or inhibition of the Calvin-cycle due to a blocked trans
location of photosynthetic products lead to this limited reoxi
dation of NADPH
z
. These conditions result in the men
tioned over-reduction of the electron transport chain and a
lack of the potential of charge separation after chlorophyll
activation in the light. Thus, inhibited energy transfer sup
ports photodynamic reactions of type II generating singlet
oxygen (Elstner et al., 1985):
Chi Chl* (Chl* = activated chlorophyll)
Chl* 3Chl eChl =triplet state chlorophyll)
3Chl+O
z
Chl+10
Z
Oxygen Activation during Plant Stress 255
The electrophilic nature of singlet oxygen results in chemi
cal reactions with double bonds or other electron rich func
tionality. Singlet oxygen may eventuelly be quenched by
carotenoides or a-tocopherol in the thylakoid membranes or
directly react with unsaturated fatty acids (LH) forming
hydroperoxides (LOOH) (Knox and Dodge, 1985):
LH + 10
2
LOOH
Membrane disruption due to lipid peroxidation is a com
mon feature of photodynamic actions. Valenzo (1987)
reviewed different photomodifications of biological mem
branes with emphasis on singlet oxygen mechanisms. He
concluded that effective membrane sensitization usually
involves an association of the sentiziser with the membrane.
This is the fact especially in thylakoid membranes of the
chloroplasts.
Limited NADP+-availability results also in the formation
0
of 0
2
and H
2
0
2
via over-reduction of electrontransport -
system and subsequent electron channelling to oxygen. Oxy
gen may be reduced by either the primary acceptor (PA) of
photosystem I, by reduced ferredoxin (fd
red
), or by both (Oss
wald and Elstner, 1986):
0
fd
red
+ O
2
fd
ox
+ 0
2

0
fd
red
+ 0
2
- + 2H+ fd
ox
+ H
2
0
Z
0
PAred + O
2
PAox + 0
2

H
2
0
2
may be further reduced by reduced ferredoxin form
ing oxygen species with properties similar to the hydroxylrad
ical (OH.) (Elstner etOal., 1985):
Light dependent generation of reactive oxygen species in
the thylakoid membrane is summerized in fig. 5.
Reductively and photodynamically generated reactive oxy
gen species degradate fatty acids in the membranes. The
formed hydroperoxides of fatty acids decay metal catalyzed
forming alkoxylradicals (LO), which in turn cooxidize
chlorophyll and other pigments, initiating their bleaching
(Elstner et aI., 1985):
LOOH+Me LO+OH-+Me
ox
LO.+Chl
red
LOH+Chl
ox
Reactive oxygen species can be detoxified in the chloro
plasts. Superoxide dismutation is catalyzed by copper zinc
SOD, which is found in the stroma as well as bound to the
thylakoids. Since chloroplasts are devoid of catalase, H
2
0
2
detoxification is brought about the ascorbate (AscH
2
/Asc)
and glutathione (2G5H/GS5G) redox cycle at the expense of
NADPH
2
<Beck-Halliwell-Asada cycle, c.f. Elstner et al.,
1985):
H
Z
0
2
+ AscH
2
Ascorbate petDxidase Asc+ 2H
2
0
Asc+2GSH AscH
2
+GSSG
GSSG + NADPH
2
Glutathione reductase 2GSH+NADP
The whole process not only detoxifies H
2
0
2
but also
decreases the NADPH:lATP ratio. Thus, lipid peroxidation
and pigment cooxidation due to the generation of reactive
oxygen species only take place when the defence system of
the chloroplast is overcharged. Furthermore accumulation of
H
2
0
2
in the stroma inhibits certain Calvin-cycle enzymes
(generally the phosphatases).
Uncoupling of oxidative phosphorylation results in insuffi
cient reoxidation of NADPH
2
and thus yields inhibition of
photosynthesis in the light due to a lack of ATP.
4.201 50
2
and derivatives
Plant damage, caused by 50
2
is oxygen and light depend
ent. Typical 50
r
effects are the rapid inhibition of photosyn
thesis before direct symptoms such as bleaching of chloro
phyll can be observed. S02 or its aqueous solution, sulfite
(HS0
3
-) can be oxidized in the presence of oxygen via a rad
ical chain mechanism (Peiser and Yang, 1978):
0
Initiation: SO/- +0
2
S03-+0
2

Progapation: S03- +0
2
S05
S05- +SO/- SOr +SO/
S04-+SO/- SO/- +S03
SOr+OH- SO/-+OH
2
OHo+S0
3
- OH-+S0
3
0
Termination: OHo+S0
3
0- OH-+S0
3
S03-+02-+2H+ S03+H
2
0
2
Ozo-+020-+2H+ H
2
0
2
+0
2
The initiation can be started by metal ions, UV-irradiation
or enzymatic reactions. Free radicals generated during sulfite
autoxidation attack cellular membranes via lipid peroxidation
and subsequent chlorophyll bleaching. In addition lipid per
oxidation is initiated by sulfite radicals generated during the
reaction between sulfite and superoxide (Peiser and Yang,
1978):
0
SO/- + 0
2
- + 2H+ S03- + H
2
0
2
S03- + LH HS0
3
- +L
Lo+0
2
LOo
LOOo+LH LOOH+Lo
The bleaching of chlorophyll by lipid peroxidation is only
observed, ifH50
3
- is simultaneously present. Neither LOOH
nor H50
3
- alone can destroy the green color of chlorophyll .
The generated H
2
0
2
is thought to be responsible for the
inactivation of ascorbate peroxidase, glutathione reductase
and enzymes of the Calvin-cycle, such as phosphatases. S02
inhibits the translocation of photosynthetic products via a
disturbed phloem loading.
4.2.2 Ozone
A disturbed phloem loading is also observed after ozone
exposure of higher plants and is associated with an inactiva
tion of the plasmalemma bound ATPase (Dominy and
Heath, I985). Secondary events lead to an increased starch
accumulation and finally bleaching of the photosynthetic
pigments.
256 SUSANNE HIPPELI and ERICH E ELSTNER
hlCV hxJ'
rourADP. I

PSII
e
--r-""'7...::....'\0::::----... PSI
"t)
ADP + P; ATP \ \ Fig. 5: Light-dependent generation of reac
tive oxygen species in the thylakoid mem
+ 2H' 02-.02-. 2W H202 + 02
brane (after Osswald and Elstner, 1986).
Ozone further acts as an effective uncoupler of the photo
phosphorylation (Robinson and Wellburn, 1983). Studies
with isolated chloroplasts showed, that ozone blockes the
energy transfer from the primary light acceptors to the reac
tion centers, initiating photodynamic reactions of type II as
mentioned above. The significance of this observations
remains to be established in vivo, since ozone can probably
not penetrate the outer chloroplast membrane.
Aben and coworkers (1990) showed that low level ozone
(120llg/m3 for 8 hours per day over two weeks) can have a
direct effect on the stomata and photosynthetic system, caus
ing decreased photosynthesis and stomatal conductance.
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