PHYSIOLOGIA PLANTARUM 118: 47–56.

2003 Copyright # Physiologia Plantarum 2003

Printed in Denmark – all rights reserved ISSN 0031-9317

Atrazine resistance entails a limited xanthophyll cycle activity, a lower

PSII efficiency and an altered pattern of excess excitation dissipation
Gyula Váradia,b, Hilda Polyánkaa, Éva Darkó1,a and Endre Lehoczkia,*
a
Department of Botany, University of Szeged, Szeged, Egyetem u. 2., H-6722, Hungary
b
Research Institute for Viticulture and Enology, Kecskeme´t, Úrihegy 5/a, H-6000, Hungary
1
Permanent address: Agricultural Research Institute of the Hungarian Academy of Sciences, Martonvásár, Hungary
*Corresponding author, e-mail: lehoczki@bio.u-szeged.hu
Received 24 July 2002; revised 25 October 2002

Atrazine-resistant (AR) weeds have a modified D1 protein
structure, with a Ser264!Gly mutation on the D1 protein,
near the plastoquinone binding niche. The photosynthetic per-
formance, the light response of the xanthophyll cycle and
chlorophyll fluorescence quenching-related parameters were
compared in attached leaves of susceptible (S) and AR bio-
types of the C3 dicot Chenopodium album L., Epilobium
adenocaulon Hausskn., Erigeron canadensis L., Senecio
vulgaris L. and Solanum nigrum L. and the C4 dicot Amaranthus
retroflexus L. grown under natural high-light conditions.
No significant difference in CO2 assimilation rate per leaf
area unit was found between the S and AR biotypes of the
investigated C3 plants, whereas the AR biotype of
A. retroflexus exhibited a relatively poor photosynthetic
performance. The D1 protein mutant plants expressed a
reduced activity of light-stimulated zeaxanthin formation.
Neither the lower violaxanthin de-epoxidase activity nor the
depletion of ascorbate seems to be the cause of the lower in
vivo zeaxanthin formation in the AR plants. All the D1
mutant weeds had limited light-induced non-photochemical
(NPQ) and photochemical (qP) quenching capacities, and dis-
played a higher photosensitivity, as characterized by the ratio
(1-qP)/NPQ and a higher susceptibility to photoinhibition.
Analysis of the chlorophyll fluorescence parameters
showed that a lower proportion of excitation energy was
allocated to PSII photochemistry, while a higher excess
of excitation remained in the AR weeds relative to the S
plants.

Introduction
Atrazine resistance in weeds is due to a mutation of the
psbA gene protein (Hirschberg et al. 1984, Gressel 1985),
which results in a Ser264!Gly substitution in the photo-
system II (PSII) reaction centre D1 protein, causing
decreased binding of atrazine and the secondary quinone
acceptor (QB) (Pfister and Arntzen 1979). Additionally,
atrazine resistance is accompanied by pleiotropic effects,
including both structural and functional changes in the
PSII complexes in the chloroplasts of atrazine-resistant
(AR) biotypes (Bowes et al. 1980, Gressel 1985, Jansen
and Pfister 1990). Numerous studies of the growth, physi-
ology and competition of weeds that are resistant or
susceptible (S) to triazine herbicides have confirmed
that in many cases the D1 protein mutation has negative
physiological consequences at the whole-plant level.
Despite research on triazine resistance in non- and iso-
nuclear biotypes, questions remain as concerns the
mechanism by which mutation affects the whole-plant
performance. AR biotypes are generally less productive
than S biotypes, especially when the plants are grown at
high-light levels (Warwick and Black 1981, Hart and
Stemler 1990) and/or at elevated temperatures (McCloskey

Abbreviations – A, antheraxanthin; AR, atrazine-resistant; D, fraction of light absorbed in PSII antennae that is thermally dissipated; Fm,
maximum fluorescence in dark-adapted leaves; Fm0 , maximum fluorescence in illuminated leaves; Fo, initial fluorescence in dark-adapted
leaves; Fo0 , chlorophyll fluorescence immediately after termination of actinic illumination; Fv ¼ Fm–Fo or Fv0 ¼ Fm0 –Fo0 , variable chlorophyll
fluorescence; Fv/Fm or Fv0 /Fm0 , quantum efficiency of charge separation in PSII; NPQ, non-photochemical fluorescence quenching; P, fraction
of light absorbed in PSII antennae that is utilized in photosynthetic electron transport; PPFD, photosynthetically active photon flux density;
qP, photochemical quenching coefficient; S, susceptible; V, violaxanthin; VDE, violaxanthin de-epoxidase; Z, zeaxanthin; X, ultimate excess of
light absorbed by PSII antennae.

Physiol. Plant. 118, 2003 47

and Holt 1991). It seems that resistance to triazine
herbicides is accompanied by a higher photosensitivity,
and thus a greater susceptibility to photoinhibition (Hart
and Stemler 1990, Curwiel et al. 1993, Sundby et al.
1993, Váradi et al. 1994a, Darkó et al. 1996, 2000) and
to high temperature (Ducruet and Lemoine 1985,
Ducruet and Ort 1988, Havaux 1989, Dulai et al. 1998),
which lowers the productivity of the AR biotype. However,
studies with AR and S weed biotypes have not always con-
firmed these conclusions (van Oorschot and Leeuwen 1984).
Oxygenic photosynthetic organisms have evolved
multiple photoprotective mechanisms to cope with the
potentially damaging effects of light and oxidative stress.
The xanthophyll cycle is known to be one of the main
photoprotective mechanisms in photosynthesizing higher
plant cells (Demmig et al. 1987, Demmig-Adams and
Adams 1992, Pfündel and Bilger 1994, Yamamoto and
Bassi 1996). As yet, there is no agreement on the
mechanism of the energy dissipation processes, but it is
presumed that, directly or indirectly, the xanthophyll
cycle may play an important role (reviewed by Demmig-
Adams and Adams 1992, Pfündel and Bilger
1994, Horton et al. 1996, Eskling et al. 1997, Gilmore
1997). A build-up of trans-thylakoid DpH along the
light-driven photosynthetic electron transport chain, the
formation of zeaxanthin (Z) and antheraxanthin (A)
from violaxanthin (V) by violaxanthin de-epoxidase
(VDE) in the chloroplast lumen, the presence of the
PsbS protein in the LHCII and a low pH-induced pro-
tonation and conformational change in the antenna are
essential for the NPQ process in higher plants, as has
been thoroughly studied mainly in artificially generated
deletion mutants of Arabidopsis thaliana (Gilmore et al.
1998, Li et al. 2000, 2002a, Müller et al. 2001).
A reduced activity of light-stimulated formation of Z
in a naturally occurring D1 mutant of a higher plant
species, the AR biotype of Conyza (Erigeron) canadensis
Cronq. (horseweed) was first revealed by Váradi et al.
(1994a), this phenomenon was accompanied by a higher
photosensitivity and sensitivity to photoinhibition
(Darkó et al. 1996, 2000). Moreover, a higher turnover
of the D1 protein has been found in the AR biotype of
Brassica napus (Sundby et al. 1993) and indirectly in AR
biotypes of E. canadensis through use of an inhibitor of
chloroplast-encoded protein synthesis (Darkó et al.
2000). These observations suggest a higher contribution
of the D1 turnover, instead of the xanthophyll cycle, to
the dissipation of excessive absorbed energy from the
photosynthetic apparatus in the D1 protein mutants. It
seems that these natural mutants might be useful model
systems for studies of the photoprotective mechanisms,
but to date they have been somewhat neglected.
Our aim is to understand the overall accompanying
mechanism related to atrazine resistance, which results in
a lowered ecological performance of the AR biotypes. In
the work reported here, we carried out investigations in
several dicot weed species on how the photosynthetic
functions are influenced by the D1 protein mutation
causing triazine resistance, and examined the general
48
occurrence of the negatively altered xanthophyll cycle
function in AR biotypes of these dicot weeds, compared
the CO2 assimilation and photosensitivity characteristics,
and assessed the allocation of the absorbed photosynthet-
ically active light energy to photochemistry and dissipa-
tion in AR and S dicot weeds.
Materials and methods
Plant material
S (wild) and naturally selected AR biotypes of Amaranthus
retroflexus, Chenopodium album, Epilobium adenocaulon,
E. canadensis, Senecio vulgaris and Solanum nigrum were
grown in well-watered pots under natural high-light
conditions in the Botanical Garden of the University of
Szeged (Hungary). Fully developed young leaves of 2- to
3-month-old plants were used for the measurements
(plants in the rosette stage were used in the case of
E. canadensis). The plants were moved to the laboratory
and kept at low (about 100 mmol m2 s1) photochem-
ically active photon flux density (PPFD) for 24 h before
light treatment for xanthophyll cycle analysis, or fluores-
cence or CO2 assimilation measurements.
Light response of the xanthophyll cycle
The interconversion of the xanthophyll cycle compon-
ents was determined in leaf discs floated on tap water
and illuminated at different PPFDs between 50 and
1800 mmol m2 s1 for 1 h before sampling. Tungsten
halogen lamps (2  500 W) above a water-filled tank
made of Plexiglass as heat filter were used as light
sources. Leaf disc samples were frozen in liquid nitrogen
immediately after sampling and stored at 80 C for
sample preparation and pigment analysis.
Conversion of violaxanthin to zeaxanthin in vitro
Isolated thylakoids, containing 1 mg ml1 chlorophyll,
were used for measurements of the conversion of V to A
and Z by VDE. For the enzymatic activity of VDE, two
preconditions must be fulfilled: a low pH and the presence
of ascorbate (Hager and Holocher 1994). Therefore, in
each test tube, 1 ml thylakoid suspension was diluted in
4 ml citrate buffer (100 mM citric acid, 200 mM K2HPO4)
at pH 5.2, and the assays of V conversion were started by
the addition of ascorbate (final concentration 30 mM).
This reaction mixture was incubated in the dark for
1 h at 26 C. The conversion was stopped by the addition
of 100 mg NaHCO3, a saturating amount of NaCl and
2–3 ml diethyl ether. Pigments were then transferred to the
ether phase by shaking and the two phases were separated
by centrifugation (1000 g, 5 min). The ether, containing
the photosynthetic pigments, was next evaporated off
under a N2 gas stream. The dried samples were solubilized
in methanol and injected onto the HPLC column. The con-
trol measurements were performed at pH 7.2 in the presence
or absence of ascorbate, or at pH 5.2 without ascorbate.
Physiol. Plant. 118, 2003
Pigment analysis
Xanthophyll cycle components were determined by
means of HPLC (Váradi et al. 1992). Leaf disc samples
were ground in liquid nitrogen in Eppendorf tubes and
extracted overnight with acetone:water (85:15) at 18 C.
After centrifugation at 9000 g, the supernatant (20 ml)
was injected directly onto the C18 RP HPLC column
(Nucleosil 120/C18/5 mm, 4.2  250 mm). All sample
preparation steps were carried out under dim-light con-
ditions. The state of interconversion of the xanthophyll
cycle components was expressed in terms of the de-epox-
idation of the xanthophyll cycle pigments, calculated as
[A 1 Z]/[V 1 A 1 Z] (Gilmore et al. 1998). Chlorophylls
and total carotenoids were measured according to
Lichtenthaler (1987).
Chlorophyll fluorescence measurements
The in vivo chlorophyll a fluorescence of wild and D1
protein mutant biotypes was traced on 20-min dark-
adapted leaves excited by a weak 583 nm measuring
light modulated at 4.8 kHz, using a Dual Channel
Modulated Fluorimeter (Hansatech, UK). The max-
imum fluorescence (Fm or Fm0 ) was induced by a white
saturating pulse (3000 mmol m2 s1, sufficient to close
all of the PSII reaction centres) of 1 s duration, provided
by a Hansatech PLS1 halogen lamp. The actinic light inten-
sity was adjusted to between 65 and 1800 mmol m2 s1
PPFD, using neutral density filters. The photochemical
fluorescence quenching coefficient (qP) and
Stern-Volmer non-photochemical quenching (NPQ) were
discriminated by applying saturating pulses every 60 s
during the last 5 min of the continuous light treatment.
After the actinic light had been switched off, far-red light
was applied for determination of the minimum level of
fluorescence at steady state (F00 ).
The photochemical quenching was calculated accord-
ing to Schreiber et al. (1986). The relative quantum yield
of PSII photochemistry, DF/Fm0 (or 1–Fs/Fm0 ), was
determined and calculated as described by Genty et al.
(1989). The index of susceptibility to light stress or
excitation pressure on PSII was defined by the quotient
(1–qP)/NPQ (Osmond 1994, Shen et al. 1996). The energy
allocation pattern of the absorbed photosynthetically
active radiation in the leaves of the wild and D1 mutant
biotypes was described according to Demmig-Adams et al.
(1996). The fraction of light absorbed in PSII antennae
that is utilized in PSII photochemistry was estimated
from P ¼ (Fv0 /Fm0 )  qP, while the fraction of dissipation
was estimated from D ¼ 1–Fv0 /Fm0 , where Fv0 is the
actual variable fluorescence (Fv0 ¼ Fm0 –F00 ) at a given
PPFD.
CO2 assimilation measurements
The light response curves of CO2 fixation were measured
in an LCA-3 infrared gas analyser (ADC, England) at
340 ppm CO2, 21% (v/v) O2, 27 C and RH ffi50%.
Physiol. Plant. 118, 2003
Plants were kept in the laboratory at about 100 mmol
m2 s1 PPFD for 24 h before the photosynthesis meas-
urements. The leaf positioned in the measuring chamber
was kept in darkness for 10 min and then illuminated by
a projector, using a heat filter (Melles Griot, Irvine, CA,
USA). Readings were taken 7 min after each step of
increasing PPFD (50–1800 mmol m2 s1).
Results
Sensitivity to photoinhibition
AR plant biotypes are known to suffer from photo-
inhibition more seriously than their S counterparts.
This is demonstrated by a more pronounced decrease in
the photochemical efficiency (Fv/Fm) of PSII, and an
increase in the constant fluorescence F0 after several
hours of photoinhibition. The time dependencies of
Fv/Fm under photoinhibitory conditions for the S
and AR biotypes of C. album, E. adenocaulon, S. vulgaris,
S. nigrum and A. retroflexus are depicted in Fig. 1.
Fv/Fm declined faster and to a higher degree for the AR
biotypes, while there was a more pronounced increase in
F0 for the AR plants (data not shown). Similar results
1.0
A B
Fv/Fm
0.5
0.0
1.0
C D
Fv/Fm
0.5
0.0
0 1 2 3 4 5 0 1 2 3 4 5
1.0
E
Fv/Fm
0.5
0.0
0 1 2 3 4 5
Time (h)
Fig. 1. Photoinhibitory conditions characterized by a gradual
decrease in efficiency of PSII (Fv/Fm) in the leaves of atrazine-
resistant (AR, open symbols) and susceptible (S, closed symbols)
biotypes of Chenopodium album (A), Epilobium adenocaulon (B),
Senecio vulgaris (C), Solanum nigrum (D) and Amaranthus
retroflexus (E). The data are means of 8 replicates from 3
independent experiments, and standard errors are visible when
larger than the symbols (for related data on Erigeron canadensis, see
Darkó et al. 1996).
49
have been reported for E. canadensis (Váradi et al. 1994a,
Darkó et al. 1996). These widely accepted parameters
demonstrating the photoinhibitory conditions in plant
leaves, however, do not reveal the photosynthetic carbon
assimilation performance or the actual efficiency of the
PSII photochemistry.
Photosynthetic performance
CO2 assimilation rates in leaves of the S and AR weed
biotypes of C. album, E. adenocaulon, S. vulgaris,
S. nigrum, E. canadensis and A. retroflexus, at different
PPFDs, are illustrated in Fig. 2. Both the rate of CO2
assimilation and the light saturation point depend on the
plant species. A. retroflexus exhibited by far the best
photosynthetic performance (i.e. the highest maximum
levels of CO2 fixation), and E. adenocaulon and
25
10
–5
Rate of CO2 assimilation per leaf area unit (µmol CO2 m–2 s–1)
25
10
–5
25
10
–5
25
10
–5
25
10
–5
25
10
–5 0
Fig. 2. Light response of the CO2 assimilation in the leaves of
atrazine-resistant (AR, open symbols) and susceptible (S, closed
symbols) biotypes of Chenopodium album (A), Epilobium
adenocaulon (B), Senecio vulgaris (C), Solanum nigrum (D),
Erigeron canadensis (E) and Amaranthus retroflexus (F) plants
measured at 340 p.p.m. CO2 and 21% (v/v) O2 ambient air
composition at 27 C and expressed relative to leaf area unit (left)
or total chlorophyll content (right). The data are means of a
minimum of 5 replicates on 3 different plants, and standard errors
are visible when larger than the symbols.
50
E. canadensis the worst. The CO2 fixation of A. retroflexus
did not reach the light saturation level expected from its C4
character, while the studied C3 dicots underwent satur-
ation in different PPFD ranges, E. adenocaulon and
E. canadensis having the lowest saturation light intensities.
With the exception of the C4 type A. retroflexus, we did
not find significant differences between the S and AR
pairs of the studied dicots in the rate of carbon fixation
on a leaf area basis (Fig. 2, left). However, there were
noteworthy differences in photosynthetic performance
for the S and AR biotypes of C. album and S. vulgaris
when the CO2 fixation was expressed relative to the total
chlorophyll content of the leaves (Fig. 2, right).
Light response of the xanthophyll cycle and photosynthetic
pigment composition
In order to obtain the light response of the xanthophyll
cycle, leaf discs floated on tap water were illuminated at
A 110 different PPFDs for 1 h. The xanthophyll cycle reached
steady-state levels within 30 min for all species, for both
Rate of CO2 assimilation per total leaf chlorophyll (µmol CO2 mmol Chl–1 s–1)
50
biotypes and at all light intensities. The xanthophyll
–10
cycle de-epoxidation processes at different PPFD levels
in the leaves of the AR and S biotypes of C. album,
B 110 E. adenocaulon, S. vulgaris, S. nigrum and A. retroflexus
plants are demonstrated in Fig. 3, expressed as the per-
50
centage pattern of the xanthophyll cycle components
–10 (left and middle) or the calculated de-epoxidation status
(right). There is a clearly retarded V de-epoxidation,
C 110
resulting in significantly reduced Z levels and a lower
50 de-epoxidation status, especially at higher PPFD values,
in the leaves of all the AR biotypes. The xanthophyll
–10 cycle of the leaves of A. retroflexus, however, displayed a
D 110 very unusual light response when investigated in floated
leaf discs. Generally, more than 50% of the xanthophyll
50 cycle pool is in the form of Z at high PPFDs in the S
(wild) biotypes, while it is less than 50% in the AR
–10
biotypes. A similar phenomenon in intact leaves in vivo
E 110 has been described for E. canadensis (Váradi et al. 1994a,
Darkó et al. 1996). Table 1 demonstrates the pigment
50 compositions of the S and AR biotypes of the investi-
gated dicot weeds. There are no clear tendencies in the
–10
total pigment contents, but the AR biotypes have a lower
chlorophyll a/b ratio. Although there are quite large
F 110 differences in the pool size of the xanthophyll cycle
between the species, especially when expressed relative
50
to the total chlorophyll content, the pool sizes do not
–10
show large and systematic differences between the two
500 1000 1500 2000 0 500 1000 1500 2000 biotypes of these weed species.
PPFD (µmol m–2 s–1) In order to clarify whether the enzymatic de-epoxidation
processes are inhibited in the AR plants the in vitro
interconversion of V to Z via A was studied at low (5.2)
or at neutral (7.2) pH in thylakoids isolated from wild and
mutant plants, in the dark, in the presence or in the
absence of ascorbate. The conversion of V to Z was
observed only at low pH and in the presence of ascorbate.
The state of de-epoxidation of the xanthophyll cycle pig-
ments was similar in the thylakoids isolated from the wild
type or the mutant biotypes (Table 2).
Physiol. Plant. 118, 2003
 100 1.0 Fig. 3. Light-induced interconversion of
A xanthophyll cycle components (left and

middle; violaxanthin, ; antheraxanthin,
,; zeaxanthin, &) and the calculated
50 0.5 de-epoxidation status ((Z 1 A)/
(V 1 A 1 Z), right; susceptible: S, closed
symbols; atrazine-resistant: AR, open
symbols) in the leaves of atrazine-resistant
0 0.0 (AR, middle) and susceptible (S, left
Xanthophyll cycle components (in % of cycle pool)

column) biotypes of Chenopodium album

(A), Epilobium adenocaulon (B), Senecio
100 1.0 vulgaris (C), Solanum nigrum (D) and
B Amaranthus retroflexus (E). The data are

De-epoxidation status (Z + A)/(V + A + Z)

means of 15 replicates from 3 independent
0.5 experiments, and standard errors are visible
50 when larger than the symbols (for related
data on Erigeron canadensis, see Váradi et al.
1994a, b; Darkó et al. 2000).
0 0.0

100 1.0
C

50 0.5

0 0.0

100 1.0
D

50 0.5

0 0.0

100 1.0
E

50 0.5

0 0.0
100 600 1100 1600 100 600 1100 1600 100 600 1100 1600

PPFD (µmol m–2 s–1)

Table 1. Pigment composition of the leaves of atrazine-resistant (AR) and susceptible (S) biotypes of Chenopodium album, Epilobium
adenocaulon, Erigeron canadensis, Senecio vulgaris, Solanum nigrum and Amaranthus retroflexus (Chl, chlorophyll; V, violaxanthin; A,
antheraxanthin; Z, zeaxanthin; n ¼ 4–5).

(V 1 A 1 Z)
Chl a + b Total carotenoids V1A1Z per Chl a + b
Species Biotype (mmol m2) Chl a/b ratio (mmol m2) (mmol m2) (mmol mmol1)
Chenopodium album S 308  44 3.22  0.08 107  6 12.4  0.6 40.2  2.1
AR 266  36 3.10  0.09 96  6 15.8  0.9 59.6  3.4
Epilobium adenocaulon S 316  37 2.98  0.05 113  9 29.0  1.1 91.8  3.8
AR 282  35 2.83  0.05 109  5 25.7  1.3 91.1  4.8
Erigeron canadensis S 364  21 3.04  0.05 103  5 17.3  2.1 47.5  4.2
AR 381  25 2.77  0.07 122  3 16.5  1.8 43.3  5.2
Senecio vulgaris S 295  23 2.72  0.01 81  7 12.9  1.4 43.7  6.2
AR 316  24 2.59  0.04 90  5 12.5  0.8 38.2  5.9
Solanum nigrum S 283  29 2.98  0.08 86  6 16.7  0.9 59.0  3.2
AR 292  32 2.88  0.05 88  6 18.2  1.3 63.2  4.6
Amaranthus retroflexus S 236  15 3.75  0.05 73  5 18.6  1.9 78.8  6.1
AR 266  11 3.50  0.05 81  5 16.7  2.1 62.8  7.8

Physiol. Plant. 118, 2003 51

Fluorescence quenching, photosensitivity and allocation of Table 2. In vitro violaxanthin de-epoxidase activity in the presence
excitation energy of ascorbate in thylakoid membranes isolated from leaves of
atrazine-resistant (AR) and susceptible (S) biotypes of Chenopodium
In order to characterize the energy dissipation processes album, Epilobium adenocaulon, Solanum nigrum and Erigeron
canadensis (V, violaxanthin; A, antheraxanthin; Z, zeaxanthin;
in the photosynthesizing leaves, chlorophyll a fluores- n ¼ 4–5).
cence data were collected by means of a modulated
measurement technique. The calculated fluorescence Species Biotype pH (A 1 Z)/(V 1 A 1 Z)
quenching parameters, NPQ, qP and the yield of PSII Chenopodium album S 7.2 0.098  0.012
photochemistry in the leaves of the AR and S biotypes of 5.2 0.495  0.040
C. album, E. adenocaulon, S. vulgaris, S. nigrum and AR 7.2 0.089  0.016
A. retroflexus plants were determined at different PPFD 5.2 0.463  0.034
levels (Fig. 4). Similar results have been reported for Epilobium adenocaulon S 7.2 0.087  0.014
E. canadensis by Darkó et al. (1996). NPQ may normally 5.2 0.495  0.035
reach a value of 2 or 3 at saturating PPFDs in the S AR 7.2 0.110  0.016
(wild) biotypes, but it remains below 2 (mainly around 1) 5.2 0.456  0.042
in the AR biotypes under similar conditions. qP declines Solanum nigrum S 7.2 0.030  0.005
5.2 0.376  0.032
as PPFD is increased. The values of qP for the AR
AR 7.2 0.050  0.004
plants, however, are always below the values for the S 5.2 0.412  0.030
plants. The yield of PSII photochemistry (Fig. 4, right)
Erigeron canadensis S 7.2 0.068  0.004
showed a characteristic light response similar to that of 5.2 0.485  0.024
qP, demonstrating a lower PSII efficiency in the AR AR 7.2 0.078  0.005
biotypes at all PPFDs. 5.2 0.496  0.026
The contributions of qP and NPQ in relation to the
photoprotection of PSII can be characterized by the ratio
(1–qP)/NPQ, which has been defined as an index of the
by mutation of the D1 protein, clearly reduces the effi-
susceptibility of PSII to light stress. The calculated light
ciency of PSII electron transport, but this may not be
sensitivity indices for leaves of the AR and S biotypes of
directly related to a lowered photosynthetic performance
C. album, E. adenocaulon, S. vulgaris, S. nigrum,
in AR plants. In accordance with van Oorschot and
E. canadensis and A. retroflexus plants are plotted against
Leeuwen (1984), we found a lower photosynthetic carbon
PPFD in Fig. 5. For the S biotypes, the light sensitivity
assimilation rate in AR A. retroflexus, whereas the rate
index seems to be nearly constant and remains at low
was comparable in the AR and S biotypes of C. album,
levels (generally between 0.1 and 0.3), whereas it
E. adenocaulon, E. canadensis, S. vulgaris and S. nigrum
approaches or sometimes exceeds 1.0 for the AR biotypes.
(Fig. 2). Differences in CO2 fixation, when expressed
Further, there is a well-defined maximum in this parameter
relative to the total chlorophyll content of the leaves,
at lower PPFDs (around 300 mmol m2 s1) for some
may indicate an altered pigment stoichiometry in the
AR biotypes, but not in the case of E. canadensis. As an
leaves of AR plants.
exception, both the AR and S biotypes of A. retroflexus
There are marked differences between the S and AR
expressed a higher photosensitivity at lower PPFDs. The
biotypes in the allocation of the absorbed light energy
light sensitivity indices for E. canadensis were calculated
to the PSII photochemistry and dissipative processes
from measurements in Darkó et al. (1996) and repeated
(Fig. 6). Modulated chlorophyll fluorescence measurements
experiments.
may furnish data of value for an assessment of the excit-
For a deeper understanding of the background and the
ation energy fraction delivered to the open PSII centres
possible consequences of the lowered xanthophyll cycle
(photochemistry) or thermally dissipated in comparisons
activity in the AR plants, the allocation of the absorbed
of wild and D1 mutant biotypes of the same species. Assess-
light energy to the PSII photochemistry and dissipative
ments were carried out according to Demmig-Adams et al.
processes was assessed for the two biotypes (Fig. 6). The
(1996), assuming matching behaviour of PSI and PSII.
fraction of light absorbed in the PSII antennae that is
Before the fluorescence measurements, plants were photo-
utilized in the PSII photochemistry was estimated from
inhibited at about 1500 mmol m2 s1 PPFD, similar to their
P ¼ (Fv0 /Fm0 )*qP, while the dissipation fraction was esti-
growing conditions. The investigated species (wild types)
mated from D ¼ 1–Fv0 /Fm0 . The investigated wild-type
showed characteristic differences in the case of the
plant species displayed quite different photochemical
S biotypes, E. canadensis and S. vulgaris having the most
efficiencies. The AR biotypes had much lower photo-
efficient PSII photochemistry, while S. nigrum might
chemical activities in all species, with a more or less
suffer worst from excessive excitation in nature. There
increased share of dissipation.
was otherwise a quite surprising variability in the parti-
tion of excessive excitation to thermal dissipation (D) and
the ultimate excess (X) that might generate reactive
Discussion
oxygen species in the antennae and/or reduce molecular
It is widely accepted that AR plant biotypes exhibit a oxygen (Demmig-Adams et al. 1996). These assessments
lowered ecological fitness. Atrazine resistance, conferred indicated that all of the studied AR biotypes exhibited

52 Physiol. Plant. 118, 2003

 4 1.0 1.0 Fig. 4. Light response of the non-
A photochemical fluorescence quenching
3 (NPQ, left), the photochemical quenching
2 0.5 0.5 (qP, middle) and the yield of PSII
photochemistry (right) in the leaves of
1 atrazine-resistant (AR, open symbols) and
0 0.0 0.0 susceptible (S, closed symbols) biotypes of
Chenopodium album (A), Epilobium
adenocaulon (B), Senecio vulgaris (C),
4 1.0 1.0 Solanum nigrum (D) and Amaranthus
3 B retroflexus (E) (for similar data on Erigeron
canadensis, see Darkó et al. 1996, 2000).

Photochemical quenching coefficient (qP)

2 0.5 0.5
Non-photochemical quenching (NPQ)

The data are means of 4 replicates from 3

Yield of PSII photochemistry (∆F/Fm')

1 independent experiments, and standard
errors are visible when larger than the
0 0.0 0.0 symbols.

4 1.0 1.0
3 C
2 0.5 0.5
1
0 0.0 0.0

4 1.0 1.0
3 D
2 0.5 0.5
1
0 0.0 0.0

4 1.0 1.0
3 E
2 0.5 0.5
1
0 0.0 0.0
0 500 100 1500 2000 0 500 100 1500 2000 0 500 100 1500 2000

PPFD (µmol m s )–2 –1

a more or less increased dissipative capacity. The most unknown special regulatory and/or dissipative processes
important fact, however, is that all of the AR biotypes that do not fully match the preconditions of this assess-
unequivocally displayed a diminished photochemical ment.
efficiency. The only well-marked strangeness in the pigment com-
There seems to be a contradiction between this indica- position (Table 1) was the consistently lower chlorophyll
tion of a sometimes much higher assessed energy dissipa- a/b ratio in the leaves of the AR plants relative to the S
tion and the lowered xanthophyll cycle activity (Fig. 3) plants for all the investigated dicots. This may indicate a
and the higher light sensitivity indices of the AR plants shift in the PSII/PSI ratio and/or (similarly as for shade-
(Fig. 5). One possible explanation for this is that AR acclimated leaves, but in contrast not for a shaded habi-
plants might have alternative defence mechanisms, e.g. tat) a relatively larger light harvesting antenna in the AR
a markedly enhanced D1 protein turnover, which may be biotypes, and this latter might result in a higher excess of
another key element of photoprotection in PSII. The excitation energy in the photosynthetic apparatus of
question remains, however, of whether the assessment these D1 protein mutants. This ‘shade-type character’,
of the allocation of the excessive excitation to thermal together with a weak xanthophyll cycle performance,
dissipation is fully adaptable to the AR biotype or not. may well explain the high photosensitivity or excitation
The calculated light sensitivity index works well with the pressure (Fig. 5) in the AR biotypes of some species, even
wild (S) biotypes and also indicates the higher photo- at low PPFDs. At all PPFDs, floated leaf discs (Fig. 3)
sensitivity of the AR plants. However, there is an anomal- showed an unusually high rate of de-epoxidation as
ous region at low PPFDs (around 300 mmol m2 s1) compared with the intact leaves in vivo (Váradi et al.
where some of the AR biotypes exhibit higher sensitivity 1994a, 1994b, Darkó et al. 1996). This may be a conse-
than at higher irradiances. It cannot be excluded, how- quence of the seriously reduced photosynthetic activity,
ever, that these D1 mutant plants may possess as yet due to the much lower diffusion velocities in the liquid

Physiol. Plant. 118, 2003 53

 1.5
Photosensitivity index or excitation pressure (1 – qP)/NPQ A B D D
1.0
C. album 'S' C. album 'AR'

P X
0.5
A P X
0.0

1.5 D D
C D E. adenocaulon 'S'
1.0 E. adenocaulon 'AR'

P X P
X
0.5
B
0.0

D D
1.5 X E. canadensis 'AR'

X
E F E. canadensis 'S'
1.0
P C P
0.5

0.0
D D
0 500 1000 1500 2000 0 500 1000 1500 2000 X S. vulgaris 'AR'

PPFD (µmol m–2 s–1) PPFD (µmol m–2 s–1)

S. vulgaris 'S'
X
P D P
Fig. 5. Light sensitivity indices ([1–qP]/NPQ) of the leaves of
atrazine-resistant (AR, open symbols) and susceptible (S, closed
symbols) biotypes of Chenopodium album (A), Epilobium
adenocaulon (B), Senecio vulgaris (C), Solanum nigrum (D),
Erigeron canadensis (E) and Amaranthus retroflexus (F). The data D D
are calculated from the results presented in Fig. 4, and standard S. nigrum 'AR'
errors are visible when larger than the symbols. S. nigrum 'S'

P X P X
phase and, as a consequence, the certainly higher excess E
of excitation in the leaf tissues under these conditions.
However, there was an uncontradictable shortfall in the
de-epoxidation in all the AR biotypes of the dicot weeds D D
as compared with the wild biotypes, even under these A. retroflexus 'S' A. retroflexus 'AR'
extreme circumstances, which may reflect differing avail-
ability of V in the two biotypes in vivo. P
There were no significant and unequivocal differences
P X F X
in xanthophyll cycle pool sizes between the S and AR
plants in the studied dicot species (Table 1), so the differ- Fig. 6. Allocation pattern of excitation energy absorbed in the PSII
ences between the two biotypes in the xanthophyll cycle antennae to PSII photochemistry (P ¼ (Fv0 /Fm0 )*qP, black areas),
performance and the NPQ process should be related to thermal dissipation (D ¼ 1–Fv0 /Fm0 , dark-grey areas) and the
remaining excess excitation (X, light-grey areas) in the leaves of
some regulatory element, possibly to the trans-thylakoid atrazine-resistant (AR, right) and susceptible (S, left) biotypes of
DpH formation and the PsbS dose per PSII unit (Li et al. Chenopodium album (A), Epilobium adenocaulon (B), Senecio
2002a). However, neither the psbS titre nor the extent of vulgaris (C), Solanum nigrum (D), Erigeron canadensis (E) and
Amaranthus retroflexus (F). The data are calculated from the
the light-induced DA535 absorbance change was investi- average values of the results presented in Fig. 4.
gated in these biotypes. Otherwise, even with the same
PsbS levels in the two biotypes, an altered degree of
protonation of PsbS due to restrained DpH formation The xanthophyll cycle activity (V de-epoxidation) has
under high-light conditions in the AR plants cannot be been suggested to be limited by a temporary ascorbate
excluded. Point mutations in psbS affecting the number deficit in vivo (Müller-Moulé et al. 2002). It has also
or the position of the protonatable amino acid residues been shown that the regulation of xanthophyll cycle-
in the PsbS on the luminal side, which might be critical dependent non-radiative energy dissipation in the
for the proper functioning and stability (Li et al. 2002b), pigment bed of PSII is modulated not only by lumen
can also be hypothesized, although this has not been acidification, but also by ascorbate availability (Neubauer
studied yet in these plant species. and Yamamoto 1994). The question arises of whether a

54 Physiol. Plant. 118, 2003

relative ascorbate depletion or a limited ascorbate avail-
ability may be limiting to Z formation in AR plants.
Fluorescence induction measurements have revealed
that the PSII primary electron acceptor, QA, is poised
in a more reduced state in the AR biotype as compared
with the wild type, which can be explained by an altered
QA–/QB equilibrium constant in the D1 mutant (Bowes
et al. 1980). The redox state of the plastoquinone pool
may affect the V availability (Siefermann and Yamamoto
1975), i.e. a slower electron flow from the quinone accep-
tors of PSII, resulting in a more oxidized state of the PQ
pool, may reduce the V availability. This may cause lower
levels of Z relative to the whole xanthophyll cycle pool.
The mechanism of this redox control on V availability is
as yet unknown.
The explanation of the lowered V de-epoxidation in
the AR biotypes may be hidden in the trans-thylakoid
DpH-related xanthophyll cycle regulation processes. It is
inferred that V de-epoxidation is controlled both at the
level of enzyme activity and at the level of V availability
(Pfündel et al. 1994). The two elements can be regulated
independently. The activity of the VDE bound to the
luminal side of the thylakoids probably depends on the
pH of localized proton domains in the thylakoids, and
the V availability is controlled by the (delocalized) lumen
pH, perhaps via the pH-dependent reoxidation of plasto-
quinol by the Cyt b/f complex. It has been shown for
E. canadensis (Darkó et al. 2000), and confirmed for
further dicot weeds, that the in vitro activity of VDE
does not differ in the thylakoids of the S and AR bio-
types near the optimum pH (5.2) and in the presence of
ascorbate (Table 2). The enzyme regulation and activities
in vivo, however, may be notably different from those
observed in vitro. Exactly the same in vivo light response
of the xanthophyll cycle was observed in intact attached
leaves of the S and AR biotypes of E. canadensis in the
presence of the strong artificial electron acceptor methyl-
viologen (paraquat) (Darkó et al. 2000, Váradi et al.
2000) and confirmed for E. adenocaulon too (data not
shown). These experimental findings indicate two import-
ant things: (1) the maximum in vivo VDE activity and
the V availability should become similar in the biotypes
when the natural mechanisms of lumen acidification
are disturbed by a strongly accelerated linear electron
transport and an extremely large trans-thylakoid DpH
probably arises, and (2) a relative ascorbate deficiency
might not be the cause of the weaker light response of Z
formation in the AR biotype. Hence, a lower actual VDE
activity due to the lowered photosynthetic electron trans-
port rate resulting in a reduced DpH formation appears to
be the possible explanation of the altered behaviour of the
xanthophyll cycle and the NPQ process and the higher
photosensitivity in the AR dicots. A possible role of the
PsbS protein must be investigated in relation to atrazine
resistance.
Acknowledgements – This work was supported by Hungarian
Research Fund (OTKA) grant No. T-035252.
Physiol. Plant. 118, 2003
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