Planta (1998) 207: 313±324
Divergent strategies of photoprotection in high-mountain plants
P. Streb1, W. Shang1, J. Feierabend1, R. Bligny2
1
Botanisches Institut, J.W. Goethe-UniversitaÈt, Postfach 11 19 32, D-60054 Frankfurt am Main, Germany
Laboratoire de Physiologie Cellulaire VeÂgeÂtale, DBMS, Unite de Recherche AssocieÂe au Centre National
de la Recherche Scienti®que 576 et Universite Joseph Fourier, Commissariat aÁ l'Energie Atomique,
17 rue des Martyrs, F-38054 Grenoble Cedex 9, France
2
Received: 18 March 1998 / Accepted: 7 August 1998
Abstract. Leaves of high-mountain plants were highly
resistant to photoinhibitory damage at low temperature.
The roles of di€erent photoprotective mechanisms were
compared. Mainly, the alpine species Ranunculus glacialis (L.) and Soldanella alpina were investigated
because they appeared to apply greatly divergent strategies of adaptation. The ratio of electron transport rates
of photosystem II/photosystem I measured in thylakoids
from R. glacialis did not indicate a speci®c acclimation
to high irradiance. Low rates of a chloroplast-mediated
inactivation of catalase (EC 1.11.1.6) in red light
indicated, however, that less reactive oxygen was
released by isolated chloroplasts from R. glacialis
than by chloroplasts from lowland plants. Leaves
of S. alpina and of Homogyne alpina (L.) Cass, but not
those of R. glacialis, had a very high capacity for
antioxidative protection, relative to lowland plants, as
indicated by a much higher tolerance against paraquatmediated photooxidative damage and a higher a-tocopherol content. Accordingly, ascorbate and glutathione
were strongly oxidized and already largely destroyed at
low paraquat concentrations in leaves of R. glacialis, but
were much less a€ected in leaves of S. alpina. Nonradiative dissipation of excitation energy was essential
for photoprotection of leaves of S. alpina and depended
on the operation of the xanthophyll cycle. Strong nonphotochemical quenching of chlorophyll ¯uorescence
occurred also in R. glacialis leaves at high irradiance, but
was largely independent of the presence of zeaxanthin or
antheraxanthin. For R. glacialis, photorespiration appeared to provide a strong electron sink and a most
essential means of photoprotection, even at low tem-
Abbreviations and symbols: DTT ˆ dithiothreitol; Fv/Fm ˆ ratio of variable to maximum chlorophyll a ¯uorescence; PAR ˆ
photosynthetically active radiation; PPT ˆ phosphinothricin;
QA ˆ primary electron acceptor of PSII; qN ˆ coecient of
non-photochemical quenching of ¯uorescence yield; qP ˆ coecient of photochemical quenching of ¯uorescence yield
Correspondence to: J. Feierabend;
E-mail: [email protected]; Fax: 49 (69) 79824822
perature. Application of phosphinothricin, which interferes with photorespiration by inhibition of glutamine
synthetase, caused a striking reduction of electron
transport through photosystem II and induced marked
photoinhibition at both ambient and low temperature in
leaves of R. glacialis, while S. alpina was less a€ected.
Key words: Alpine plant ± Antioxidant ± Paraquat
tolerance ± Photoinhibition ± Photorespiration ±
Xanthophyll cycle
Introduction
High-mountain plants depend on a highly ecient
carbon assimilation since their growing season is very
short. Although the extreme climatic conditions with
extended periods of high irradiance and low temperature, rapid temperature changes, and a reduced partial
pressure of CO2 are not favorable for photosynthesis,
the photosynthetic performance of alpine plants appears
to be well adapted to the environment. Previous studies
have shown that in several alpine plants photosynthesis
is highly ecient at cool temperatures and also adapted
to high irradiance (Moser et al. 1977; KoÈrner and
Larcher 1988). Highly sensitive and widespread symptoms of photodamage in non-adapted plants are the
photoinactivation of photosystem II (PSII) and of the
peroxisomal enzyme catalase. The reaction-centre protein D1 of PSII and catalase usually have a rapid
turnover in light and therefore require continuous repair
by new synthesis. (Hertwig et al. 1992; Aro et al. 1993,
Foyer et al. 1994; Feierabend et al. 1996). In nonadapted plants the apparent loss of both PSII and
catalase activity by photoinactivation is greatly enhanced at low temperature where protein synthesis is
suppressed. (Feierabend et al. 1992; Huner et al. 1993;
Wise 1995). However, in leaves of the alpine plants,
Homogyne alpina, Soldanella alpina and Ranunculus
glacialis, PSII and catalase are highly resistant to
314
photoinhibitory damage even when protein synthesis is
suppressed by inhibitors of translation or by exposure to
low temperature (Streb et al. 1997). The results of
previous investigations (Streb et al. 1997) suggested that
the maintenance of stable levels of the D1 protein of
PSII (Shang and Feierabend 1998) and of catalase did
not greatly depend on repair by new protein synthesis.
However, in most instances this high stability was only
observed in intact leaves. In vitro, both PSII of isolated
thylakoids from H. alpina and R. glacialis and catalase
in cell-free leaf extracts from S. alpina and R. glacialis
were as rapidly photoinactivated as known for lowelevation plants. The only exception was a catalase from
H. alpina which after extraction also had a much higher
stability in light than other catalases (Streb et al. 1997).
In all other instances the high resistance to photoinactivation of PSII and catalase must depend on the
presence of more ecient mechanisms of protection
against oxidative damage in leaves of the alpine plants
than in non-adapted low-elevation plants.
Unexpectedly, the strategies of protection appear to
be very divergent in the alpine plants H. alpina,
R. glacialis and S. alpina (Streb et al. 1997). Important
mechanisms for the avoidance of reactive-oxygen production in chloroplasts, or for its removal, are the
thermal dissipation of excitation energy which has
frequently been correlated with the light-dependent
formation of zeaxanthin in the xanthophyll cycle
(Demmig-Adams and Adams III 1996), superoxide
dismutase, and antioxidants and antioxidative enzymes
of the ascorbate-glutathione cycle (Asada 1994; Foyer
et al. 1994). Accordingly, high contents of xanthophyllcycle pigments, of the antioxidants ascorbate and
glutathione, and of antioxidative enzymes have usually
been observed in cold-adapted plants (SchoÈner and
Krause 1990; Wise 1995; Thiele et al. 1996; Leipner et al.
1997), and the levels of these compounds and enzymes
are also extraordinarily high in leaves of the alpine
plant S. alpina but not in those of H. alpina and
R. glacialis (Wildi and LuÈtz 1996; Streb et al. 1997).
While the occurrence of a light-stable catalase might
contribute to the light-stress tolerance of H. alpina, the
very poor antioxidative systems in R. glacialis are
unusual. Therefore, we have now further evaluated the
relevance of various protective mechanisms to the lightstress tolerance of alpine plants and mainly concentrated
on a comparison of the two most divergent species
S. alpina and R. glacialis. Where appropriate, properties
of the alpine plants were compared with those of
lowland plants that were either collected from ®eld sites
(Ranunculus, Taraxacum) or grow under controlled
laboratory conditions.
Chloroplasts were analysed for potential functional
adaptations. In those plants that allowed the isolation of
intact organelles in sucient yields. The overall strength
of antioxidative protection was examined by a comparison of the sensitivity of the leaves to paraquat which
mediates superoxide formation by electron transfer from
PSI to O2 and thus greatly enhances photooxidative
damage (Dodge 1994). The role of the xanthophyll-cycle
pigments was analysed by application of dithiothreitol
P. Streb et al: Photoprotection in alpine plants
(DTT) which inhibits the formation of zeaxanthin from
violaxanthin (Demmig-Adams et al. 1990). Further, the
herbicide phosphinothricin (PPT) was applied as a tool
to assess the role of photorespiration. Phosphinothricin
irreversibly inhibits glutamine synthetase and prevents
the reassimilation of ammonia. As a result, photorespiration is suppressed because glutamate is lacking for the
transamination of glyoxylate to glycine (Wendler and
Wild 1990; Wendler et al. 1990; Kozaki and Takeba
1996). Photorespiration appears to provide an essential
mechanism to protect the photosynthetic apparatus
against photooxidative damage under strong irradiance
(Wu et al. 1991; Heber et al. 1996; Kozaki and Takeba
1996).
Materials and methods
Plant materials and ®eld sites. Alpine plants were investigated
during the months June and July in the years 1996 and 1997 in the
laboratories of the Station Alpine du Lautaret of the University
Joseph Fourier of Grenoble at the Col du Lautaret (2100 m
altitude) in the western part of the French Alps. Leaves of
Soldanella alpina L., Homogyne alpina (L.) Cass and Ranunculus
glacialis L. were collected from the same ®eld sites at 2400±2700 m
between the Lautaret and the Galibier pass that were described by
Streb et al. (1997). Mean daily temperatures and light intensities
during the investigation period were as previously reported (Dorne
et al. 1986; Streb et al 1997). For comparison, fully expanded
mature leaves of the non-alpine plants Taraxacum ocinale
Wiggers and Ranunculus acris L. were collected from unshaded
sites of the Botanical Garden of the University of Frankfurt am
Main during the months July till October. Some experiments were
performed with primary leaves of rye plants (Secale cereale L. cv.
Halo) grown under constant conditions either for 6 d at 22 °C or
for 5 weeks at 4 °C and a photon ¯ux of 96 lmol m)2 s)1
photosynthetically active radiation (PAR) in the presence of a
modi®ed nutrient solution, as previously described (Streb et al.
1993).
Experimental treatments and light exposure. For experimental
treatments under controlled conditions, incident light of 500±
2000 lmol m)2 s)1 PAR was generated with halogen lamps (20 W,
75 W and 100 W). The intensity of sunlight was not constant
during the incubation periods. Incident light varied between 650
and 2350 lmol m)2 s)1 PAR, with a mean photon ¯ux of
1800 lmol m)2 s)1 PAR. Periods of incident light of lower than
1500 lmol m)2 s)1 PAR were short, resulting from a few clouds
during the course of the experiments. The ambient temperature
during the incubations in sunlight ranged from 10 °C to 27 °C with
a mean temperature of 18 °C. When incubations were performed in
an ice bath, the temperature observed in sunlight at the leaf surface
ranged between 4 °C and 9 °C, with a mean temperature of 7 °C.
For incubations in sunlight, di€erential treatments were always
performed in parallel, in order to exclude e€ects caused by
variations of light or temperature conditions that might occur on
di€erent days of exposure.
For incubations in the presence of paraquat, leaves were
collected in the morning. Leaves were ¯oated in Petri dishes on
water or paraquat solution and irradiated for 24 h under temperature-controlled conditions at 25 °C with white light of 1000 lmol
m)2 s)1 PAR. For experiments with DTT, leaves were collected in
the evening and incubated overnight for approximately 14 h at
20 °C in darkness in Petri dishes on water (controls) or solutions of
3 mM or 30 mM DTT. For experiments with PPT, leaves were
collected in the morning and incubated for 2 h in Petri dishes on
water or solutions of 1 mM PPT at an ambient temperature of
approximately 20 °C in weak room light. Subsequent to the latter
P. Streb et al: Photoprotection in alpine plants
preincubation periods, leaves treated with DTT or PPT and
respective controls were exposed to constant light conditions
between 500 and 2000 lmol m)2 s)1 PAR at either 25 °C or on ice,
or to natural sunlight at either ambient temperature or on ice.
Isolation of chloroplasts. For the isolation of chloroplasts, leaves
were cut to small pieces and ®nely minced with razor blades under
ice-cold conditions, using the grinding medium of pH 6.1 described
by Jensen and Bassham (1966), except that KNO3 was omitted and
10 mM Na2S2O4 was used as reducing agent. Alternatively,
homogenates without Na2S2O4 were prepared in a glove bag under
an N2 atmosphere. The homogenate was passed through four
layers of muslin and one layer of Miracloth (Calbiochem) and
loaded onto stepped Percoll gradients according to HoÈinghaus and
Feierabend (1983). The gradients consisted of layers of 15%, 42%,
and 80% (v/v) Percoll contained in a bu€er medium of 0.3 M
sucrose, 50 mM Mes-KOH (pH 6.1), 5 mM Na2EDTA, 5 mM
MnCl2, 5 mM MgCl2, and were centrifuged for 5 min at 10 000 g
and 4 °C. Intact chloroplasts were collected from the interface of
42% and 80% Percoll. The chloroplast fractions were diluted 2- to
3-fold either with the assay medium for chloroplast-mediated
catalase inactivation, consisting of 0.33 M sorbitol, 50 mM HepesKOH (pH 7.6), 2 mM Na2EDTA, 1 mM MgCl2, 10 mM KCl or
with assay medium used for the estimation of photosynthetic
electron transport activities. Chloroplasts were re-pelleted by 5 min
centrifugation at 4000 g and 4 °C, re-suspended with the respective
assay medium and adjusted to a chlorophyll concentration of 1 mg
ml)1. Intactness of chloroplasts was examined according to Heber
and Santarius (1970) and amounted, on average, to 83%.
Chloroplast-mediated catalase inactivation. For the assay of chloroplast-mediated in vitro inactivation of catalase, puri®ed bovine
liver catalase (10 lkat ml)1, Sigma) was incubated together with a
suspension of Percoll-gradient-puri®ed intact chloroplasts (50 lg
chlorophyll ml)1) in the assay medium used for the re-suspension of
the chloroplasts. The mixture was incubated in a water bath at
25 °C in red light (800 lmol photons m)2 s)1 PAR) provided by
Osram halogen lamps in combination with the Schott ®lters RG 1
(>50% transmission above 610 nm) and KG 3.
Analytical methods. Electron transport rates were measured at
25 °C with a Hansatech (Kings Lynn, Norfolk, UK) oxygen
electrode at saturating light intensity. For the assays the concentrated chloroplast fractions were diluted with H2O to a ®nal
chlorophyll concentration of 30 lg ml)1. The PSII electron transport was assayed with phenyl-p-benzoquinone as acceptor according to Hundal et al. (1995). The PSI electron transport from
ascorbate to methyl viologen and whole-chain electron transport
from H2O to methyl viologen were assayed according to Allen and
Holmes (1986). All assays contained 5 mM NH4Cl.
Leaves were extracted by the preparation of homogenates of
5.0 ml ®nal volume from 0.4 g leaf tissue (fresh weight). Homogenates for catalase (EC 1.11.1.6) measurements were prepared in
50 mM K-phosphate bu€er (pH 7.5) at 4 °C, and enzyme activity
was estimated according to Streb et al. (1993).
Homogenates for glutamine synthetase (EC 6.3.1.2) measurements were prepared according to Nesselhut and Harnischfeger
(1981) in 50 mM imidazole bu€er (pH 7.5) containing 15 mM
MgSO4, 8 mM 2-mercaptoethanol and 10 mM ATP. The extracts
were centrifuged for 30 min at 4 °C and 37 000 g and supernatants
were used for the enzyme assays. Glutamine synthetase activity was
assayed as the hydroxylamine-dependent synthetase reaction
according to Nesselhut and Harnischfeger (1981). An extinction
coecient of e ˆ 0.6043 mM)1 cm)1 was determined at 535 nm
for the Fe-glutamylhydroxamate complex.
Antioxidants were extracted in 1% (w/v) metaphosphoric acid
and analysed by HPLC as described previously (Streb and
Feierabend 1996), except that an Eurosphere-100-C18 reversedphase column (Knauer, Berlin, Germany) was used for the
separation of ascorbic acid and glutathione. The total chlorophyll
content was estimated according to Arnon (1949) in 80% acetone
315
extracts. For analysis by HPLC of pigments and a-tocopherol,
approximately 0.4 g of fresh leaf tissue was frozen in liquid
nitrogen and stored at )20 °C until use. The leaf material was
extracted, essentially as described by Thayer and BjoÈrkman (1990).
Frozen leaves were ground with mortar and pestle in liquid
nitrogen in the presence of some CaCO3 and extracted in 1 ml of
99% (v/v) acetone in darkness for 15 min under a nitrogen
atmosphere at room temperature. The extract was centrifuged for
12 min at 4000 g. Extraction and centrifugation were repeated and
the resulting supernatants combined and ®ltered through a 0.45 lm
Nalgene ®lter. Xanthophyll-cycle pigments and chlorophylls were
separated and quanti®ed by HPLC, as described previously (Streb
et al. 1997), except that a non-endcapped LiChrosphere-100-C18
reversed-phase column from Knauer was used. In HPLC separations of pigment extracts, a-tocopherol was detected and quanti®ed
with a ¯uorescence detector (excitation at 295 nm; emission at
330 nm), as described by SchuÈep and Rettenmaier (1994). Contents
of xanthophyll-cycle pigments and a-tocopherol were expressed on
the basis of total chlorophyll content, as determined in the
separation of the same sample.
Light-dependent oxygen evolution of leaves was measured with
a Hansatech (Kings Lynn, Norfolk, UK) leaf-disc electrode at a
saturating photon ¯ux in the presence of 5±10% CO2, as described
by Streb et al. (1997). Chlorophyll a ¯uorescence was measured
with a portable photosynthesis yield analyser (Mini-Pam; H. Walz,
E€eltrich, Germany). Initial ¯uorescence (Fo) was determined after
excitation of a dark-adapted leaf with weak modulated light in the
ML-burst mode of the Mini-Pam to con®rm that the measuring
light caused no closure of PSII reaction centres. Maximum
¯uorescence (Fm) was measured after a saturating light pulse of
approximately 7000 lmol m)2 s)1 PAR. Actinic light of 500±
2000 lmol m)2 s)1 PAR was provided by an external halogen
lamp in combination with a ventilated infrared ®lter (2060-M,
20 W; H. Walz). The leaf temperature was maintained between
22 °C and 28 °C. For measurements at low temperature, leaves
were kept on an ice-cold support. For irradiation with 500 lmol
m)2 s)1 PAR a cold light source (Schott KL 1500) with two glass®bre leads was used.The leaf temperature was 4 °C. Saturating
light pulses of 2 s duration were applied every 2 min during
exposure to actinic light or darkness. Photoinhibition of PSII was
determined as decrease of the ratio of variable to maximum
chlorophyll a ¯uorescence (Fv/Fm) after a minimum of 10 min
dark adaptation (Krause and Weis 1991). In some experiments Fv/
Fm was measured after dark periods of up to 1 h. Approximations
of the electron transport rate through PSII were calculated as
(Fm¢)F)/Fm¢ ´ PAR ´ 0.5 ´ 0.8 by multiplying the quantum eciency of PSII according to Genty et al. (1989) by the incident
photon ¯ux density of PAR and an average factor of 0.8 for leaf
absorptance, and dividing by a factor of 2 to account for the
sharing of absorbed photons between the two photosystems. In
this equation, F is the actual and Fm¢ the corresponding maximum
¯uorescence in light. The photochemical quenching (qP) and the
non-photochemical quenching (qN) were calculated according to
Schreiber et al. (1986) at the end of the actinic light irradiations
and corrected for quenching of Fo according to Bilger and
Schreiber (1986). The approximate percentage of reduced primary
electron acceptor of PSII (QA) was calculated as 1)qP according to
Dietz et al. (1985).
All experiments were repeated independently at least three
times. Mean values with standard errors of the mean are presented.
Results
Functional properties of chloroplasts. Of the three alpine
plant species, Homogyne alpina, Ranunculus glacialis and
Soldanella alpina, that were used for our investigations,
only R. glacialis allowed the isolation of chloroplasts
with high yields. Therefore, only this plant was used for
316
Fig. 1. Comparison of the activities of PSII, PSI and whole-chain
electron transport in isolated thylakoid membranes obtained from
leaves of R. glacialis and R. acris. For PSII-mediated electron
transport, phenyl-p-benzoquinone (PBQ) was used as electron
acceptor; PSI was assayed with ascorbate (Asc) as electron donor
and methyl viologen (MV ) as acceptor; for whole-chain electron
transport (PSII + PSI), methyl viologen was used as acceptor
a comparison of photochemical activities in vitro. The
activities of PSI and PSII and whole-chain electron
transport were assayed with thylakoids obtained
from Percoll-gradient-puri®ed intact chloroplasts from
R. glacialis and compared with corresponding preparations from the lowland plant R. acris (Fig. 1). The in
vitro activities of both photosystems were more than
50% higher in R. glacialis than in R. acris, and the
capacity of whole-chain electron transport was almost
twice as high. The ratio of PSII/PSI was not, however,
signi®cantly di€erent in the two plant species, and did
not therefore indicate any peculiar high-light acclimation in the alpine species. The ratio of measured electron
transport rates of PSII/PSI amounted to 1.6 for
R. glacialis and to 1.4 for R. acris.
Previous investigations have indicated that the inactivation of the light-sensitive enzyme catalase in vitro is
not only mediated by blue light absorbed by its heme
prosthetic groups but may also occur as a result of
photooxidative events initiated in chloroplasts when the
enzyme is irradiated in a suspension together with the
latter organelle (Feierabend and Engel 1986; Feierabend
et al. 1996). Chloroplast-mediated inactivation of catalase is best demonstrated by irradiation of a mixture of
intact chloroplasts and catalase with red light which is
not absorbed by the catalase and did not inactivate the
enzyme (Fig. 2). Rates of catalase inactivation were very
similar in the presence of intact chloroplasts from the
lowland plants Taraxacum ocinale or Secale cereale.
According to additional investigations, chloroplast-mediated inactivation was only observed when appropriate
electron acceptors for the photosynthetic electron transport were missing, especially when the plastoquinone
compounds were not oxidized. Under such conditions
reactive oxygen species, including superoxide, are generated and mediate the oxidative inactivation of catalase
(data not shown). When intact isolated chloroplasts
P. Streb et al: Photoprotection in alpine plants
Fig. 2. Comparison of the inactivation of bovine liver catalase in the
presence of intact isolated chloroplasts from leaves of S. cereale (e),
T. ocinale (h), and R. glacialis (n) in red light. s, control with
catalase in red light in the absence of chloroplasts; m, control with
catalase and isolated chloroplasts from R. glacialis in darkness; n,
control with catalase and isolated chloroplasts from T. ocinale in
darkness
Table 1. Comparison of the contents of a-tocopherol in the leaves
of the alpine plants R. glacialis and S. alpina and in the primary
leaves of S. cereale. Leaves of alpine plants were collected from
®eld sites, S. cereale was grown under controlled conditions for 6 d
at 22 °C (non-hardened) or for 5 weeks at 4 °C (cold-hardened) in
96 lmol m)2 s)1 PAR continuous white light. The leaves of the
alpine plants were exposed to sunlight for 3 h before extraction
Plant species
a-Tocopherol
[mmol (mol chlorophyll))1]
S. cereale, 22 °C-grown
S. cereale, 4 °C-grown
R. glacialis
S. alpina
18.9
38.7
4.3
271.8
‹
‹
‹
‹
1.6
2.3
3.6
57.6
from R. glacialis were irradiated together with catalase
in red light, only very little inactivation of catalase was
observed. This result suggests that R. glacialis chloroplasts had an improved ability to avoid the accumulation of reactive oxygen species that were detected
through the inactivation of accompanying catalase.
The content of a-tocopherol which represents the main
scavenger for free radicals in thylakoid membranes was,
however, extraordinarily low in leaves of R. glacialis
(Table 1). While the content of a-tocopherol greatly
increased when S. cereale leaves were cold-hardened by
growing the plants at 4 °C, the amount of a-tocopherol
in R. glacialis leaves accounted for only about 1.5% of
that found in S. alpina leaves and was even much lower
than in non-hardened S. cereale leaves grown in low
light at 22 °C.
Paraquat sensitivity. In order to assess and compare the
capacities for antioxidative protection, paraquat tolerance was compared in leaves of the alpine plants
H. alpina, R. glacialis and S. alpina and in those of
the lowland plants R. acris and T. ocinale. As
symptoms of oxidative damage, the bleaching of chlorophyll and the inactivation of catalase (see Streb et al.
1993) were measured at di€erent concentrations of
P. Streb et al: Photoprotection in alpine plants
Fig. 3. Catalase activity and chlorophyll content (as percent of
control without paraquat) in leaves of the alpine plants S. alpina (s),
H. alpina (,), R. glacialis (h) and the lowland plants T. ocinale (r)
and R. acris (m) after 24 h exposure to light of 1000 lmol m)2 s)1
PAR at 25 °C in the presence of di€erent paraquat concentrations
paraquat (Fig. 3). In general, catalase was more sensitive
to paraquat-induced photooxidation than chlorophyll.
A striking di€erence in paraquat-sensitivity was observed between the lowland plants R. acris and
T. ocinale and the alpine species H. alpina and
S. alpina. In the lowland plants, catalase activity was
totally lost in the presence of 10 lM paraquat and in
T. ocinale chlorophyll was also largely degraded at this
concentration. In R. acris chlorophyll had a somewhat
higher stability in the presence of paraquat. The alpine
species H. alpina and S. alpina exhibited a much higher
paraquat tolerance, with respect to photooxidation of
both catalase and chlorophyll, than the lowland plants.
At 100 lM paraquat they had still retained more than
50% of their chlorophyll contents. However, the alpine
species R. glacialis was as sensitive to paraquat-induced
photooxidation as the lowland plants. Chlorophyll
degradation was even more paraquat-sensitive in
R. glacialis than in R. acris (Fig. 3).
In leaves of two alpine species which greatly di€ered
in their paraquat-tolerance, changes in the contents of
the antioxidants ascorbate and glutathione were analyzed after incubation at di€erent paraquat concentrations in the light (Fig. 4). Incubation of the leaves in
light without paraquat did not greatly a€ect the antioxidant contents, except for a slight increase in the content
of total glutathione (GSH and GSSG, the reduced and
oxidized forms, respectively) in R. glacialis. With
increasing paraquat concentration, total ascorbate
(reduced ascorbate + dehydroascorbate) as well as total
glutathione (GSH + GSSG) were greatly degraded.
However, this degradation occurred at a much lower
317
Fig. 4. Comparison of the e€ects of di€erent paraquat concentrations
on the contents of reduced ascorbate (AA ) and dehydroascorbate
(DHA ), and of reduced (GSH ) and oxidized (GSSG ) glutathione in
leaves of S. alpina and R. glacialis. Leaves were assayed before
(untreated control) and after a 24-h exposure to light of 1000 lmol
m)2 s)1 PAR at 25 °C in the presence of paraquat. Di€erent scales are
used for the antioxidant contents of R. glacialis and S. alpina
paraquat concentration in R. glacialis than in S. alpina.
Thus the ascorbate content of S. alpina at 100 lM
paraquat was still as high as in R. glacialis leaves in the
absence of paraquat. In addition, both ascorbate and
GSH were already almost totally oxidized at paraquat
concentrations as low as 2 lM in R. glacialis, whereas
in S. alpina leaves the percentage of reduced ascorbate
was, even at 100 lM, higher than that of its oxidized
form (Fig. 4).
E€ects of inhibitors interfering with zeaxanthin formation
and photorespiration. The application of DTT has been
introduced to inhibit the light-induced de-epoxidation of
violaxanthin and was found to suppress non-photochemical ¯uorescence quenching and thermal dissipation
of excitation energy which often appeared to be
correlated with the occurrence of zeaxanthin (DemmigAdams et al. 1990; Demmig-Adams and Adams III
1996). After 14 h overnight incubation at 20 °C in
darkness, zeaxanthin was not detectable in leaves of
R. glacialis but a considerable amount (13% of the total
xanthophyll-cycle carotenoids) was still present in
S. alpina leaves. Zeaxanthin and antheraxanthin together amounted to approximately 30% of the xanthophyllcycle pigments in S. alpina leaves after 14 h dark
incubation. After exposure to light the contents of both
zeaxanthin and antheraxanthin increased strongly in
318
P. Streb et al: Photoprotection in alpine plants
Fig. 5. Comparison of the contents of zeaxanthin, antheraxanthin
and violaxanthin in leaves of S. alpina and R. glacialis after treatment
with DTT. After a 14-h preincubation in darkness (Dark), in the
absence (Control) or presence of 30 mM DTT, leaves were exposed
for 10 min to 1000 lmol m)2 s)1 PAR light at 25 °C or for 3 h to
sunlight either at ambient temperature or on ice
R. glacialis as well as in S. alpina. Within 10 min
exposure to strong light the de-epoxidation of violaxanthin had essentially reached its maximum (Fig. 5).
However, also in light, both the amount of zeaxanthin
per unit chlorophyll and its proportion among the total
xanthophyll-cycle pigments remained much lower in
R. glacialis than in S. alpina, as pointed out previously
(Streb et al. 1997). In addition, the total contents of
xanthophyll-cycle pigments declined during the light
exposure in R. glacialis. Incubation of leaves in the
presence of 30 mM DTT during the light exposures
prevented the formation of zeaxanthin in R. glacialis and
blocked its additional accumulation in S. alpina. A
lower concentration of 3 mM DTT, which is usually
applied in experiments, was not sucient to suppress
zeaxanthin formation in these plants. The contents of
zeaxanthin and antheraxanthin that were still present at
the end of the dark period in S. alpina declined during
the exposure to light in the presence of DTT, but
zeaxanthin could not be totally depleted in these leaves.
Particularly high levels of zeaxanthin and antheraxanthin were retained in the presence of DTT when the
leaves were kept on ice during irradiation with sunlight
(Fig. 5).
At low light intensities the non-photochemical
quenching of chlorophyll ¯uorescence (qN) was considerably higher in S. alpina than in R. glacialis. In
both plants it increased with the photon ¯ux to a
similar maximum level (Fig. 6). This dose-dependent
Fig. 6. In¯uence of DTT on the reduction state of the primary
electron acceptor of PSII QA (1)qP), non-photochemical ¯uorescence
quenching (qN), and on the calculated electron transport rates
through PSII in leaves of S. alpina (s, d) or R. glacialis (h, n)
exposed to di€erent light intensities in the absence (open symbols) or
presence (®lled symbols) of 30 mM DTT. Measurements were
performed at the end of a 10-min (500±1500 lmol m)2 s)1 PAR) or
30-min (2000 lmol m)2 s)1) irradiation with actinic light following a
14-h preincubation in darkness at 20 °C in the absence or presence of
30 mM DTT
increase of qN in light was diminished in both plant
species in the presence of DTT; however, at 2000 lmol
m)2 s)1 light in the presence of DTT, qN was ®nally
higher in R. glacialis than in S. alpina. In S. alpina
the proportion of reduced QA, indicated as 1)qP,
strongly increased and the calculated rate of electron
transport through PSII declined in the presence of
DTT at both low and high photon ¯ux. In R. glacialis
the primary acceptor QA was under all conditions in a
more oxidized state than in S. alpina. In untreated
control leaves the proportion of reduced QA was only
about half as high as in S. alpina. Vice versa, the
electron transport rate through PSII was always much
higher in R. glacialis than in S. alpina and increased
with the photon ¯ux up to 2000 lmol m)2 s)1 PAR
(Fig. 6). After treatment with DTT increases in the
proportion of reduced QA and inhibition of the
electron transport through PSII were seen only at
P. Streb et al: Photoprotection in alpine plants
319
Fig. 8. Changes in the Fv/Fm ratios in leaves of R. glacialis and
S. alpina during a 3-h exposure to sunlight at ambient temperature or
on ice in the absence or presence of either 30 mM DTT or 1 mM
PPT. The light exposures were preceded by a 14-h preincubation in
darkness at 20 °C on water or with 30 mM DTT, or a 2-h
preincubation in weak room light at 20 °C with 1 mM PPT. Control,
measurements taken before exposure to sunlight. Fv/Fm ratios were
determined after 10 min dark adaptation
Fig. 7. Comparison of the e€ects of DTT and PPT at low
temperature on non-photochemical ¯uorescence quenching (qN), the
calculated electron transport rates through PSII, the reduction state of
the primary electron acceptor QA (1)qP), and the Fv/Fm ratios in
leaves of S. alpina and R. glacialis. Leaves were preincubated at 20 °C
for 14 h in darkness with 30 mM DTT or for 2 h with 1 mM PPT in
weak room light before the 30-min exposure to actinic light of
500 lmol m)2 s)1 PAR on ice (4±7 °C). Controls were kept on water.
For determinations of qN, electron transport and (1)qP), measurements were performed at the end of the 30-min irradiation period. For
Fv/Fm the change induced by the 30-min irradiation is indicated.
Measurements of Fv/Fm at the end of the light period were performed
after an additional 10 or 30 min dark adaptation in order to
document potential recovery in darkness
higher light intensities exceeding 1000 lmol m)2 s)1
PAR in R. glacialis.
When the leaves were kept at a low temperature on
ice at a photon ¯ux of 500 lmol m)2 s)1 PAR, which
allowed di€erences in QA reduction to be recognized,
both the reduction state of QA and qN were higher than
at 25 °C in R. glacialis as well as in S. alpina, while the
electron transport through PSII was strongly retarded.
In the presence of DTT the electron transport rates and
qN were reduced and the reduction state of QA further
increased at low temperature (Fig. 7). These changes
were similar in both species, except that the electron
transport rate through PSII was also at low temperature
under all experimental conditions several-fold higher in
R. glacialis than in S. alpina. During short irradiation
periods, as applied in the experiments of Figs. 6 and 7,
the Fv/Fm ratio was not yet markedly a€ected by the
presence of DTT at 500 lmol m)2 s)1 PAR and low
temperature (Fig. 7). After 30 min at 2000 lmol m)2 s)1
and 25 °C a more marked decline of 31% was induced
by the application of DTT in S. alpina, while the decline
in R. glacialis was only 14% (data not shown). After a
3-h exposure to sunlight the di€erential e€ects of DTT
became more apparent (Fig. 8). In sunlight the decline
of Fv/Fm was greatly enhanced in S. alpina when the
leaves were incubated on ice or in the presence of DTT,
while the latter treatments had only minor e€ects in
R. glacialis, when applied separately. Only when low
temperature and DTT were applied simultaneously was
there a more marked 28% decline of the Fv/Fm ratio also
in R. glacialis. This was, however, still much smaller
than the 60% decline observed under these conditions
in S. alpina (Fig. 8).
In order to suppress photorespiration, leaves were
treated with PPT. After a 2-h preincubation with PPT at
low-intensity room light, leaves of R. glacialis had lost
85%, and leaves of S. alpina 90% of their glutamine
synthetase activity. In PPT-treated leaves photosynthetic
oxygen evolution in the presence of saturating CO2 was
between 80±90% of the rate measured in untreated
leaves, demonstrating that the inhibitor exerted no
major direct e€ects on photosynthesis under non-photorespiratory conditions (Table 2). During exposure to
1000 lmol m)2 s)1 PAR in the presence of PPT the
proportions of reduced QA and qN were increased. The
relative extent of these changes was much larger in
320
P. Streb et al: Photoprotection in alpine plants
Table 2. E€ect of a 2-h treatment with 1 mM PPT on glutamine
synthetase activity and on total photosynthetic oxygen evolution
(at saturating CO2) in leaves of R. glacialis and S. alpina. One mg
chlorophyll is equivalent to a leaf area of 2.2 ´ 10)3 m2 for
R. glacialis and of 2.7 ´ 10)3 m2 for S. alpina
Plant species
Glutamine
synthetase
[nkat (g FW))1]
Oxygen evolution
[lmol h)1 (mg
chlorophyll))1]
R. glacialis
)PPT
+PPT
68.6 ‹ 8.7
10.3 ‹ 4.8
251.1 ‹ 22.6
222.6 ‹ 21.9
S. alpina
)PPT
+PPT
57.3 + 6.3
5.5 + 1.7
199.6 ‹ 13.4
163.1 ‹ 19.6
R. glacialis than in S. alpina (Fig. 9), and at 25 °C than
at low temperature (Fig. 7). The most striking e€ect of
PPT was that it strongly reduced the electron transport
rate through PSII in R. glacialis but not in S. alpina
(Fig. 9). In R. glacialis the electron transport rate further
declined during the light exposure in the presence of PPT
but was only slightly a€ected in S. alpina.
Application of PPT induced a decline in the Fv/Fm
ratio in R. glacialis as well as in S. alpina leaves when
they were irradiated with 1000 lmol m)2 s)1 PAR at
ambient temperature (Fig. 9). During the next approximately 30 min of a subsequent dark incubation a slow
recovery from photoinhibition was observed in both
plant species. Prolonged exposure to sunlight in the
presence of PPT induced marked declines in the Fv/Fm
ratio of about 50% within 3 h in both R. glacialis and
S. alpina. Similar declines in the Fv/Fm ratio were also
seen when treated leaves were, in addition, kept on ice.
However, while in S. alpina the presence of PPT did
not much further enhance the extent of photoinhibition
that was also observed in its absence on ice, the decline
in the Fv/Fm ratio seen in ice-incubated R. glacialis
leaves was mainly induced by the presence of PPT
(Fig. 8). The rate of light-saturated photosynthetic
oxygen evolution in the presence of saturating CO2
also declined during exposure to PPT in light. The
decline in photosynthetic oxygen evolution, in general,
slightly exceeded the decline in the Fv/Fm ratio (data
not shown).
Discussion
Chilling in the light is harmful to green plants because
the reoxidation of the photosynthetic electron transport
chain, as indicated by the redox state of QA (Havaux
1987; OÈquist et al. 1993), is impaired, photooxidative
reactions are enhanced (Wise 1995) and the repair of
damaged proteins is suppressed. Photoinactivation of
PSII and of catalase are particularly sensitive symptoms
of photodamage that have been observed in many plants
at chilling temperatures (OÈquist et al. 1987; Feierabend
et al. 1992; Huner et al. 1993). However, in the leaves of
three alpine high-mountain plants that were previously
investigated, PSII as well as catalase remained fairly
stable at high irradiance and low temperature, even
when translation was blocked by inhibitors, although
Fig. 9A,B. Comparison of the e€ects of
PPT on the reduction state of the primary
electron acceptor QA (1)qP) of PSII, nonphotochemical ¯uorescence quenching
(qN), the calculated electron transport rates
through PSII and the Fv/Fm ratios in leaves
of R. glacialis (h, n) and S. alpina (s, d)
during a 60-min exposure to actinic light of
1000 lmol m)2 s)1 PAR at 25 °C. Leaves
were preincubated for 2 h in weak room
light at 20 °C in the presence of 1 mM PPT
or water (for controls) before the 60-min
high light exposure. A Measurements of
1-qP and qN were performed at the end of
the 60-min light exposure. B Electron
transport rates were estimated and calculated every 10 min during the light exposure. Open symbols, controls without PPT;
®lled symbols, incubations with PPT. The
Fv/Fm ratios were determined before and at
the end of the 60-min light exposure and
during a subsequent 1-h dark incubation, in
order to document potential recovery in
darkness
P. Streb et al: Photoprotection in alpine plants
both PSII and catalase activity could be readily photoinactivated after extraction from the leaves. An exception was that H. alpina contained a more stable catalase
(Streb et al. 1997). Ecient means for the avoidance of,
or protection from, photoinhibitory damage may be
provided by the thermal dissipation of excess excitation
energy, scavenger systems for the removal of reactive
oxygen species, and photorespiration which may serve as
a sink for the consumption of excess reducing equivalents (Asada 1994; Foyer et al. 1994). The capacities and
the functional relevance of di€erent protective systems
have now been compared in high-mountain plants,
mainly in S. alpina and R. glacialis.
Dissipation of excess absorbed light energy as heat by
non-photochemical quenching of excited antenna chlorophyll depends on the generation of a transthylakoid
proton gradient by photosynthetic electron transport
and was found to be mostly correlated with the
operation of the xanthophyll cycle and the light-dependent formation of zeaxanthin and antheraxanthin
(Demmig-Adams and Adams 1996). The relevance of
zeaxanthin formation for non-photochemical quenching
and photoprotection is, however, still controversial,
since the violaxanthin-to-zeaxanthin conversion did
not correlate with the time course of heat emission
(Havaux and Tardy 1997) and in Chlamydomonas
mutants zeaxanthin formation was neither a necessary
precondition for non-photochemical quenching nor
required for survival in excessive light (Niyogi et al.
1997a). In addition, lutein also appeared to contribute to
non-photochemical quenching and to the dissipation of
excess absorbed light energy (Niyogi et al. 1997b).
In S. alpina thermal energy dissipation by non-photochemical quenching mechanisms, potentially related to
xanthophyll-cycle operation, appeared to be important
for light stress tolerance, as reported for other coldacclimated plants (SchoÈner and Krause 1990; Adams et
al. 1995; Haldimann et al. 1996; Leipner et al. 1997).
Although the capacity for non-photochemical ¯uorescence quenching of R. glacialis was also high, it was only
to a minor extent related to the presence of zeaxanthin
and antheraxanthin. In leaves of S. alpina the zeaxanthin and antheraxanthin contents were much higher
than in those of R. glacialis, and a substantial amount of
zeaxanthin (+antheraxanthin) was retained overnight
even after extended dark periods at 20 °C. Previously,
the retention of high zeaxanthin levels in the dark had
been observed after very cold nights in overwintering
leaves and correlated with a decrease in the Fv/Fm ratio
(Adams et al. 1995). Similarly, leaves of S. alpina that
had survived over the winter always had a slightly
reduced Fv/Fm ratio between 0.6 and 0.7 (Fig. 8),
indicating that a weak chronic photoinhibition (Streb
et al. 1997), and a high level of qN (Fig. 6) already
accompanied the retention of zeaxanthin at moderate
irradiance. Even though the pre-existing zeaxanthin
could not be totally depleted, the inhibition of the
additional light-induced violaxanthin-to-zeaxanthin
conversion by DTT prevented the photon-¯ux-dependent increase of qN, greatly increased the proportion of
reduced QA, and induced marked photoinhibition of
321
PSII in sunlight in S. alpina, thus emphasizing the
signi®cance of a xanthophyll-cycle-related energy dissipation for this plant. By contrast, in R. glacialis the
e€ects of DTT application on photosynthetic functions
were much less marked. Whereas R. glacialis leaves
remained totally depleted of zeaxanthin after dark
incubation and in the presence of DTT, non-radiative
excitation energy dissipation (qN) was, nevertheless,
greatly increasing with the photon ¯ux and appeared to
be mostly independent of the operation of the xanthophyll cycle. That this zeaxanthin-independent excitation
energy dissipation also contributed to photoprotection
was suggested by the fact that the Fv/Fm ratio was only
little a€ected when R. glacialis leaves were treated with
DTT at ambient temperature. Only when exposed to
DTT under chilling conditions, did the proportion of
reduced QA increase more markedly (Fig. 7) and the Fv/
Fm ratio decline in sunlight (Fig. 8) also in R. glacialis,
although to a much lesser extent than in S. alpina.
The striking di€erences of paraquat tolerance
between R. glacialis, which behaved like a non-acclimated plant, and the other alpine plants, S. alpina and
H. alpina, provided compelling evidence that previously
observed large di€erences in the activities of antioxidative enzymes and of the contents of the antioxidants
ascorbate and glutathione existing in these species (Streb
et al. 1997) were indeed of substantial functional
signi®cance. It is unlikely that the di€erences were
related to major limitations of paraquat uptake since the
application of PPT was e€ective within 2 h in both plant
species (Table 2) and substantial degradation of antioxidants indicated the presence of paraquat also in leaves
of S. alpina. The low eciency of the reactive oxygenscavenger systems of R. glacialis leaves was further
accentuated by their low a-tocopherol content and by
the strong oxidation and destruction of ascorbate and
glutathione at low paraquat levels, although this is very
exceptional among cold-acclimated plants. Usually high
levels of antioxidative enzymes and antioxidants, as
occurring in S. alpina, were observed in cold-acclimated
leaves and regarded as essential for low-temperature
acclimation (SchoÈner and Krause 1990; Wise 1995;
Leipner et al. 1997).
Photosynthetic performance of R. glacialis was most
markedly a€ected by PPT. Phosphinothricin inhibits
glutamine synthetase and blocks the reassimilation of
ammonia which appears to be a rate-limiting step for
photorespiration (Kozaki and Takeba 1996). Photosynthesis is inhibited after PPT application, however, only
under photorespiratory conditions. Detailed investigations have shown that the inhibition of photosynthesis
was largely restored by the application of glutamine and
did not result from toxic e€ects of ammonia accumulation but was mainly related to the repression of
photorespiration (Wendler and Wild 1990; Wendler
et al. 1990). In transgenic tobacco plants in which the
plastidic glutamine synthetase was either suppressed or
overexpressed, the capacity for photorespiration changed with the level of expression of this enzyme.
The resistance of these plants against photoinhibition
of PSII and photooxidative bleaching increased in
322
proportion to the capacities of glutamine synthetase and
photorespiration, thus emphasizing the protective role
of the latter (Kozaki and Takeba 1996). Photorespiratory production of CO2 may sustain the operation of
the Calvin cycle under unfavorable conditions and serve
as a means for the dissipation of excess excitation
energy. Based on the above-mentioned results and
conclusions, the very striking reduction of the apparent
electron transport rates through PSII in R. glacialis,
which was quite similar to that occurring in transgenic
plants with reduced glutamine synthetase activity (Kozaki and Takeba 1996), indicates that photorespiration
provided a major electron sink for R. glacialis, but
appeared to be of minor relevance for S. alpina.
Athough qN increased in the presence of PPT, this was
obviously not sucient to prevent QA becoming more
strongly reduced and photoinhibition of PSII becoming
enhanced also in R. glacialis. Surprisingly, PPT was also
e€ective at low temperature. While in S. alpina the low
temperature induced some photoinhibition that was not
much further enhanced by PPT, in R. glacialis a major
decline in the Fv/Fm ratio was only observed when PPT
was present under chilling conditions (Fig. 8). While
photorespiration was mostly not expected to play a
major role at low temperature (Wise 1995; Hurry et al.
1996; Streb et al. 1997), our present results suggest that
in R. glacialis it may serve as a most essential
photoprotective mechanism also at low temperature
(Figs. 7, 8). Because of the low partial pressure of CO2,
photorespiration may be of particular importance for
some high-mountain plants. It is very unlikely that a
de®ciency of amino acids and the resulting insucient
protein synthesis caused the photoinhibition induced by
PPT, since PSII did not greatly depend on protein
synthesis in these alpine plants (Streb et al. 1997; Shang
and Feierabend 1998), and at low temperature, repair
cannot have been of major importance within the short
time periods.
In conclusion, our comparative assessment of photoprotective mechanisms revealed greatly divergent strategies in two high-mountain plants. While S. alpina, like
other cold-acclimated plants, was characterized not only
by greatly enhanced capacities of scavenger systems for
superoxide and H2O2 but also by high levels of
xanthophyll cycle pigments which already correlated
with strong non-photochemical quenching at relatively
low light intensities, this was not the case in R. glacialis.
Leaves of R. glacialis sustained very high rates of
electron transport, and this was in accord with high
in-vitro activities of both photosystems, as compared to
the low-elevation species R. acris, and with a high rate of
photosynthetic oxygen evolution (Streb et al. 1997).
However, in contrast to leaves of other plants acclimated
to high irradiance (Anderson et al. 1995), the ratio of
measured electron transport activities of PSII/PSI was
not signi®cantly increased and the chlorophyll a/b ratio
(2.7) was relatively low (Streb et al. 1997). Since QA was
generally maintained in a highly oxidized state, strong
electron sinks must be present in R. glacialis leaves also
at low temperature. Consequently, non-radiative dissipation of excitation energy gained importance only at
P. Streb et al: Photoprotection in alpine plants
higher light intensities. The low capacities of the
ascorbate-glutathione cycle and of the thylakoid-bound
radical scavenger a-tocopherol, which were con®rmed
by the high paraquat sensitivity, exclude the possibility
that the Mehler reaction might serve as major electron
sink in R. glacialis under natural conditions, because the
leaves would have su€ered from severe photooxidative
damage. Provided that no unknown alternative electron
sink was available, the leaves of R. glacialis must
maintain a highly ecient photosynthetic carbon metabolism that warrants the prompt consumption of
reducing equivalents even under unfavorable conditions.
Evidence has been presented that an increased capacity
for carbon assimilation enables cold-hardened winter
cereals to keep QA oxidized and to avoid photoinhibition (Huner et al. 1993), whereas photorespiration
appears to be insigni®cant for these plants at low
temperature (Hurry et al. 1996). Our present results
suggest that photorespiration did provide a major
electron sink for R. glacialis and was also essential for
its photoprotection at low temperature; this possibility,
however, needs to be further demonstrated by direct
measurements.
Additional adaptive mechanisms which are independent of carbon assimilation or photorespiration must,
however, exist in the chloroplasts of R. glacialis. When
isolated intact chloroplasts were irradiated without an
electron acceptor in vitro the appearance of PSII
photoinhibition was signi®cantly delayed, relative to
broken chloroplasts (Streb et al. 1997). Furthermore, the
oxidative inactivation of catalase in red light, a process
which is mediated by isolated chloroplasts from lowland
plants and provides a means to detect the release of
reactive oxygen by this organelle, was much lower in the
presence of chloroplasts from R. glacialis. These chloroplasts must therefore be able to avoid the production
of those reactive oxygens that were deleterious for
catalase, or to detoxify them more eciently than
chloroplasts from lowland plants. The latter possibility
is, however, less likely because the content of the radical
scavenger a-tocopherol was extremely low in R. glacialis. Only the concentrations of ascorbate and glutathione
were relatively high within the chloroplasts from
R. glacialis, as compared to non-alpine plants (Streb
et al. 1997), but not sucient to improve the general
paraquat tolerance of the leaves.
We thank the Deutsche Forschungsgemeinschaft, Bonn, for
®nancial support. Travel grants by the Dr. Senckenbergische
Stiftung and the Herrmann Willkomm-Stiftung, Frankfurt am
Main, are greatly appreciated. P.S., W.Sh. and J.F. are very
grateful for the great hospitality and support which they received at
the Station Alpine du Lautaret, Universite Joseph Fourier, in
France. Phosphinothricin was kindly supplied by the AgrEvo
GmbH, Frankfurt am Main.
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