Atrazine resistance entails a limited xanthophyll cycle activity, a lower

PSII efficiency and an altered pattern of excess excitation dissipation

Gyula Varadia,b, Hilda Polyankaa, Eva Darko1,a and Endre Lehoczkia,*

aDepartment of Botany, University of Szeged, Szeged, Egyetem u. 2., H-6722, HungarybResearch Institute for Viticulture and Enology, Kecskemet, Urihegy 5/a, H-6000, Hungary1Permanent address: Agricultural Research Institute of the Hungarian Academy of Sciences, Martonvasar, 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 werecompared in attached leaves of susceptible (S) and AR bio-

types of the C3 dicot Chenopodium album L., Epilobiumadenocaulon Hausskn., Erigeron canadensis L., Seneciovulgaris L. and Solanum nigrum L. and the C4 dicot Amaranthusretroflexus 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 ofA. 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 D1mutant 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 Splants.

Introduction

Atrazine resistance in weeds is due to a mutation of thepsbA 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, causingdecreased binding of atrazine and the secondary quinoneacceptor (QB) (Pfister and Arntzen 1979). Additionally,atrazine resistance is accompanied by pleiotropic effects,including both structural and functional changes in thePSII complexes in the chloroplasts of atrazine-resistant(AR) biotypes (Bowes et al. 1980, Gressel 1985, Jansenand Pfister 1990). Numerous studies of the growth, physi-

ology and competition of weeds that are resistant orsusceptible (S) to triazine herbicides have confirmedthat in many cases the D1 protein mutation has negativephysiological consequences at the whole-plant level.Despite research on triazine resistance in non- and iso-nuclear biotypes, questions remain as concerns themechanism by which mutation affects the whole-plantperformance. AR biotypes are generally less productivethan S biotypes, especially when the plants are grown athigh-light levels (Warwick and Black 1981, Hart andStemler 1990) and/or at elevated temperatures (McCloskey

PHYSIOLOGIA PLANTARUM 118: 47–56. 2003 Copyright# Physiologia Plantarum 2003

Printed in Denmark – all rights reserved ISSN 0031-9317

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; Fm

0, maximum fluorescence in illuminated leaves; Fo, initial fluorescence in dark-adaptedleaves; Fo

0, chlorophyll fluorescence immediately after termination of actinic illumination; Fv¼Fm–Fo or Fv0 ¼Fm

0–Fo0, variable chlorophyll

fluorescence; Fv/Fm or Fv0/Fm

0, quantum efficiency of charge separation in PSII; NPQ, non-photochemical fluorescence quenching; P, fractionof 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 oflight absorbed by PSII antennae.

Physiol. Plant. 118, 2003 47

and Holt 1991). It seems that resistance to triazineherbicides is accompanied by a higher photosensitivity,and thus a greater susceptibility to photoinhibition (Hartand Stemler 1990, Curwiel et al. 1993, Sundby et al.1993, Varadi et al. 1994a, Darko et al. 1996, 2000) andto 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 thepotentially damaging effects of light and oxidative stress.The xanthophyll cycle is known to be one of the mainphotoprotective mechanisms in photosynthesizing higherplant cells (Demmig et al. 1987, Demmig-Adams andAdams 1992, Pfundel and Bilger 1994, Yamamoto andBassi 1996). As yet, there is no agreement on themechanism of the energy dissipation processes, but it ispresumed that, directly or indirectly, the xanthophyllcycle may play an important role (reviewed by Demmig-Adams and Adams 1992, Pfundel and Bilger1994, Horton et al. 1996, Eskling et al. 1997, Gilmore1997). A build-up of trans-thylakoid DpH along thelight-driven photosynthetic electron transport chain, theformation of zeaxanthin (Z) and antheraxanthin (A)from violaxanthin (V) by violaxanthin de-epoxidase(VDE) in the chloroplast lumen, the presence of thePsbS protein in the LHCII and a low pH-induced pro-tonation and conformational change in the antenna areessential for the NPQ process in higher plants, as hasbeen thoroughly studied mainly in artificially generateddeletion mutants of Arabidopsis thaliana (Gilmore et al.1998, Li et al. 2000, 2002a, Muller et al. 2001).A reduced activity of light-stimulated formation of Z

in a naturally occurring D1 mutant of a higher plantspecies, the AR biotype of Conyza (Erigeron) canadensisCronq. (horseweed) was first revealed by Varadi et al.(1994a), this phenomenon was accompanied by a higherphotosensitivity and sensitivity to photoinhibition(Darko et al. 1996, 2000). Moreover, a higher turnoverof the D1 protein has been found in the AR biotype ofBrassica napus (Sundby et al. 1993) and indirectly in ARbiotypes of E. canadensis through use of an inhibitor ofchloroplast-encoded protein synthesis (Darko et al.2000). These observations suggest a higher contributionof the D1 turnover, instead of the xanthophyll cycle, tothe dissipation of excessive absorbed energy from thephotosynthetic apparatus in the D1 protein mutants. Itseems that these natural mutants might be useful modelsystems 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 ina lowered ecological performance of the AR biotypes. Inthe work reported here, we carried out investigations inseveral dicot weed species on how the photosyntheticfunctions are influenced by the D1 protein mutationcausing triazine resistance, and examined the general

occurrence of the negatively altered xanthophyll cyclefunction in AR biotypes of these dicot weeds, comparedthe 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 Amaranthusretroflexus, Chenopodium album, Epilobium adenocaulon,E. canadensis, Senecio vulgaris and Solanum nigrum weregrown in well-watered pots under natural high-lightconditions in the Botanical Garden of the University ofSzeged (Hungary). Fully developed young leaves of 2- to3-month-old plants were used for the measurements(plants in the rosette stage were used in the case ofE. canadensis). The plants were moved to the laboratoryand kept at low (about 100mmolm�2 s�1) photochem-ically active photon flux density (PPFD) for 24 h beforelight 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 waterand illuminated at different PPFDs between 50 and1800mmolm�2 s�1 for 1 h before sampling. Tungstenhalogen lamps (2� 500W) above a water-filled tankmade of Plexiglass as heat filter were used as lightsources. Leaf disc samples were frozen in liquid nitrogenimmediately after sampling and stored at �80�C forsample preparation and pigment analysis.

Conversion of violaxanthin to zeaxanthin in vitro

Isolated thylakoids, containing 1mgml�1 chlorophyll,were used for measurements of the conversion of V to Aand Z by VDE. For the enzymatic activity of VDE, twopreconditions must be fulfilled: a low pH and the presenceof ascorbate (Hager and Holocher 1994). Therefore, ineach test tube, 1ml thylakoid suspension was diluted in4ml citrate buffer (100 mM citric acid, 200 mM K2HPO4)at pH5.2, and the assays of V conversion were started bythe addition of ascorbate (final concentration 30mM).This reaction mixture was incubated in the dark for1 h at 26�C. The conversion was stopped by the additionof 100mg NaHCO3, a saturating amount of NaCl and2–3ml diethyl ether. Pigments were then transferred to theether phase by shaking and the two phases were separatedby centrifugation (1000g, 5min). The ether, containingthe photosynthetic pigments, was next evaporated offunder a N2 gas stream. The dried samples were solubilizedin methanol and injected onto the HPLC column. The con-trol measurements were performed at pH7.2 in the presenceor absence of ascorbate, or at pH5.2 without ascorbate.

48 Physiol. Plant. 118, 2003

Pigment analysis

Xanthophyll cycle components were determined bymeans of HPLC (Varadi et al. 1992). Leaf disc sampleswere ground in liquid nitrogen in Eppendorf tubes andextracted overnight with acetone:water (85:15) at �18�C.After centrifugation at 9000 g, the supernatant (20ml)was injected directly onto the C18 RP HPLC column(Nucleosil 120/C18/5mm, 4.2� 250mm). All samplepreparation steps were carried out under dim-light con-ditions. The state of interconversion of the xanthophyllcycle components was expressed in terms of the de-epox-idation of the xanthophyll cycle pigments, calculated as[A1Z]/[V1A1Z] (Gilmore et al. 1998). Chlorophyllsand total carotenoids were measured according toLichtenthaler (1987).

Chlorophyll fluorescence measurements

The in vivo chlorophyll a fluorescence of wild and D1protein mutant biotypes was traced on 20-min dark-adapted leaves excited by a weak 583 nm measuringlight modulated at 4.8 kHz, using a Dual ChannelModulated Fluorimeter (Hansatech, UK). The max-imum fluorescence (Fm or Fm

0) was induced by a whitesaturating pulse (3000mmolm�2 s�1, sufficient to closeall of the PSII reaction centres) of 1 s duration, providedby a Hansatech PLS1 halogen lamp. The actinic light inten-sity was adjusted to between 65 and 1800mmolm�2s�1

PPFD, using neutral density filters. The photochemicalfluorescence quenching coefficient (qP) andStern-Volmer non-photochemical quenching (NPQ) werediscriminated by applying saturating pulses every 60 sduring the last 5min of the continuous light treatment.After the actinic light had been switched off, far-red lightwas applied for determination of the minimum level offluorescence at steady state (F0

0).The photochemical quenching was calculated accord-

ing to Schreiber et al. (1986). The relative quantum yieldof PSII photochemistry, DF/Fm

0 (or 1–Fs/Fm0), was

determined and calculated as described by Genty et al.(1989). The index of susceptibility to light stress orexcitation pressure on PSII was defined by the quotient(1–qP)/NPQ (Osmond 1994, Shen et al. 1996). The energyallocation pattern of the absorbed photosyntheticallyactive radiation in the leaves of the wild and D1 mutantbiotypes was described according to Demmig-Adams et al.(1996). The fraction of light absorbed in PSII antennaethat is utilized in PSII photochemistry was estimatedfrom P¼ (Fv

0/Fm0)� qP, while the fraction of dissipation

was estimated from D¼ 1–Fv0/Fm

0, where Fv0 is the

actual variable fluorescence (Fv0 ¼Fm

0–F00) at a given

PPFD.

CO2 assimilation measurements

The light response curves of CO2 fixation were measuredin an LCA-3 infrared gas analyser (ADC, England) at340 ppm CO2, 21% (v/v) O2, 27

�C and RH ffi50%.

Plants were kept in the laboratory at about 100 mmolm�2 s�1 PPFD for 24 h before the photosynthesis meas-urements. The leaf positioned in the measuring chamberwas kept in darkness for 10min and then illuminated bya projector, using a heat filter (Melles Griot, Irvine, CA,USA). Readings were taken 7min after each step ofincreasing PPFD (50–1800 mmolm�2 s�1).

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 inthe photochemical efficiency (Fv/Fm) of PSII, and anincrease in the constant fluorescence F0 after severalhours of photoinhibition. The time dependencies ofFv/Fm under photoinhibitory conditions for the Sand 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 ARbiotypes, while there was a more pronounced increase inF0 for the AR plants (data not shown). Similar results

A

F v/F

mF v

/Fm

F v/F

m

0.0

0.5

1.0

B

C

0 1 2 3 4 5 0 1 2 3 4 50.0

0.5

1.0

D

E

Time (h)0 1 2 3

0.0

0.5

1.0

4 5

Fig. 1. Photoinhibitory conditions characterized by a gradualdecrease 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 Amaranthusretroflexus (E). The data are means of 8 replicates from 3independent experiments, and standard errors are visible whenlarger than the symbols (for related data on Erigeron canadensis, seeDarko et al. 1996).

Physiol. Plant. 118, 2003 49

have been reported for E. canadensis (Varadi et al. 1994a,Darko et al. 1996). These widely accepted parametersdemonstrating the photoinhibitory conditions in plantleaves, however, do not reveal the photosynthetic carbonassimilation performance or the actual efficiency of thePSII photochemistry.

Photosynthetic performance

CO2 assimilation rates in leaves of the S and AR weedbiotypes of C. album, E. adenocaulon, S. vulgaris,S. nigrum, E. canadensis and A. retroflexus, at differentPPFDs, are illustrated in Fig. 2. Both the rate of CO2

assimilation and the light saturation point depend on theplant species. A. retroflexus exhibited by far the bestphotosynthetic performance (i.e. the highest maximumlevels of CO2 fixation), and E. adenocaulon and

E. canadensis the worst. The CO2 fixation of A. retroflexusdid 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 andE. canadensis having the lowest saturation light intensities.With the exception of the C4 type A. retroflexus, we didnot find significant differences between the S and ARpairs of the studied dicots in the rate of carbon fixationon a leaf area basis (Fig. 2, left). However, there werenoteworthy differences in photosynthetic performancefor the S and AR biotypes of C. album and S. vulgariswhen the CO2 fixation was expressed relative to the totalchlorophyll 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 xanthophyllcycle, leaf discs floated on tap water were illuminated atdifferent PPFDs for 1 h. The xanthophyll cycle reachedsteady-state levels within 30min for all species, for bothbiotypes and at all light intensities. The xanthophyllcycle de-epoxidation processes at different PPFD levelsin the leaves of the AR and S biotypes of C. album,E. adenocaulon, S. vulgaris, S. nigrum and A. retroflexusplants are demonstrated in Fig. 3, expressed as the per-centage pattern of the xanthophyll cycle components(left and middle) or the calculated de-epoxidation status(right). There is a clearly retarded V de-epoxidation,resulting in significantly reduced Z levels and a lowerde-epoxidation status, especially at higher PPFD values,in the leaves of all the AR biotypes. The xanthophyllcycle of the leaves of A. retroflexus, however, displayed avery unusual light response when investigated in floatedleaf discs. Generally, more than 50% of the xanthophyllcycle pool is in the form of Z at high PPFDs in the S(wild) biotypes, while it is less than 50% in the ARbiotypes. A similar phenomenon in intact leaves in vivohas been described for E. canadensis (Varadi et al. 1994a,Darko et al. 1996). Table 1 demonstrates the pigmentcompositions of the S and AR biotypes of the investi-gated dicot weeds. There are no clear tendencies in thetotal pigment contents, but the AR biotypes have a lowerchlorophyll a/b ratio. Although there are quite largedifferences in the pool size of the xanthophyll cyclebetween the species, especially when expressed relativeto the total chlorophyll content, the pool sizes do notshow large and systematic differences between the twobiotypes of these weed species.In order to clarify whether the enzymatic de-epoxidation

processes are inhibited in the AR plants the in vitrointerconversion of V to Z via A was studied at low (5.2)or at neutral (7.2) pH in thylakoids isolated from wild andmutant plants, in the dark, in the presence or in theabsence of ascorbate. The conversion of V to Z wasobserved 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 wildtype or the mutant biotypes (Table 2).

A

Rat

e of

CO

2 as

sim

ilatio

n pe

r le

af a

rea

unit

(µm

ol C

O2

m–2

s–1 )

–5

10

25

B

–5

10

25

C

–5

10

25

D

–5

10

25

F

PPFD (µmol m–2 s–1)

0 500 1000 1500 2000–5

10

25

Rat

e o

f C

O2

assi

mila

tion

per

tota

l lea

f ch

loro

phyl

l (µm

ol C

O2 m

mol

Chl

–1 s–

1 )

–10

50

110

–10

50

110

–10

50

110

–10

50

110

–10

50

110

0 500 1000 1500 2000–10

50

110

E

–5

10

25

Fig. 2. Light response of the CO2 assimilation in the leaves ofatrazine-resistant (AR, open symbols) and susceptible (S, closedsymbols) biotypes of Chenopodium album (A), Epilobiumadenocaulon (B), Senecio vulgaris (C), Solanum nigrum (D),Erigeron canadensis (E) and Amaranthus retroflexus (F) plantsmeasured at 340 p.p.m. CO2 and 21% (v/v) O2 ambient aircomposition at 27�C and expressed relative to leaf area unit (left)or total chlorophyll content (right). The data are means of aminimum of 5 replicates on 3 different plants, and standard errorsare visible when larger than the symbols.

50 Physiol. Plant. 118, 2003

0

50

100

0.0

0.5

1.0

0.0

0.5

1.0

0.0

0.5

1.0

0.0

0.5

1.0

0.0

0.5

1.0

0

50

100

Xan

thop

hyll

cycl

e co

mpo

nent

s (i

n %

of

cycl

e po

ol)

0

50

100

De-

epox

idat

ion

stat

us (

Z +

A)/

(V +

A +

Z)

100 600 1100 16000

50

100

PPFD (µmol m–2 s–1)

100 600 1100 1600 100 600 1100 1600

0

50

100

A

B

C

D

E

Fig. 3. Light-induced interconversion ofxanthophyll cycle components (left andmiddle; violaxanthin, �; antheraxanthin,,; zeaxanthin, &) and the calculatedde-epoxidation status ((Z1A)/(V1A1Z), right; susceptible: S, closedsymbols; atrazine-resistant: AR, opensymbols) in the leaves of atrazine-resistant(AR, middle) and susceptible (S, leftcolumn) biotypes of Chenopodium album(A), Epilobium adenocaulon (B), Seneciovulgaris (C), Solanum nigrum (D) andAmaranthus retroflexus (E). The data aremeans of 15 replicates from 3 independentexperiments, and standard errors are visiblewhen larger than the symbols (for relateddata on Erigeron canadensis, see Varadi et al.1994a, b; Darko et al. 2000).

Table 1. Pigment composition of the leaves of atrazine-resistant (AR) and susceptible (S) biotypes of Chenopodium album, Epilobiumadenocaulon, Erigeron canadensis, Senecio vulgaris, Solanum nigrum and Amaranthus retroflexus (Chl, chlorophyll; V, violaxanthin; A,antheraxanthin; Z, zeaxanthin; n¼ 4–5).

Species BiotypeChl a+ b(mmol m�2) Chl a/b ratio

Total carotenoids(mmol m�2)

V1A1Z(mmol m�2)

(V1A1Z)per Chl a+ b(mmol mmol�1)

Chenopodium album S 308� 44 3.22� 0.08 107� 6 12.4� 0.6 40.2� 2.1AR 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� 3.8AR 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� 4.2AR 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� 6.2AR 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� 3.2AR 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� 6.1AR 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

excitation energy

In order to characterize the energy dissipation processesin the photosynthesizing leaves, chlorophyll a fluores-cence data were collected by means of a modulatedmeasurement technique. The calculated fluorescencequenching parameters, NPQ, qP and the yield of PSIIphotochemistry in the leaves of the AR and S biotypes ofC. album, E. adenocaulon, S. vulgaris, S. nigrum andA. retroflexus plants were determined at different PPFDlevels (Fig. 4). Similar results have been reported forE. canadensis by Darko et al. (1996). NPQ may normallyreach a value of 2 or 3 at saturating PPFDs in the S(wild) biotypes, but it remains below 2 (mainly around 1)in the AR biotypes under similar conditions. qP declinesas PPFD is increased. The values of qP for the ARplants, however, are always below the values for the Splants. The yield of PSII photochemistry (Fig. 4, right)showed a characteristic light response similar to that ofqP, demonstrating a lower PSII efficiency in the ARbiotypes at all PPFDs.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 thesusceptibility of PSII to light stress. The calculated lightsensitivity indices for leaves of the AR and S biotypes ofC. album, E. adenocaulon, S. vulgaris, S. nigrum,E. canadensis and A. retroflexus plants are plotted againstPPFD in Fig. 5. For the S biotypes, the light sensitivityindex seems to be nearly constant and remains at lowlevels (generally between 0.1 and 0.3), whereas itapproaches or sometimes exceeds 1.0 for the AR biotypes.Further, there is a well-defined maximum in this parameterat lower PPFDs (around 300mmolm�2 s�1) for someAR biotypes, but not in the case of E. canadensis. As anexception, both the AR and S biotypes of A. retroflexusexpressed a higher photosensitivity at lower PPFDs. Thelight sensitivity indices for E. canadensis were calculatedfrom measurements in Darko et al. (1996) and repeatedexperiments.For a deeper understanding of the background and the

possible consequences of the lowered xanthophyll cycleactivity in the AR plants, the allocation of the absorbedlight energy to the PSII photochemistry and dissipativeprocesses was assessed for the two biotypes (Fig. 6). Thefraction of light absorbed in the PSII antennae that isutilized in the PSII photochemistry was estimated fromP ¼ (Fv

0/Fm0)*qP, while the dissipation fraction was esti-

mated from D¼ 1–Fv0/Fm

0. The investigated wild-typeplant species displayed quite different photochemicalefficiencies. The AR biotypes had much lower photo-chemical activities in all species, with a more or lessincreased share of dissipation.

Discussion

It is widely accepted that AR plant biotypes exhibit alowered ecological fitness. Atrazine resistance, conferred

by mutation of the D1 protein, clearly reduces the effi-ciency of PSII electron transport, but this may not bedirectly related to a lowered photosynthetic performancein AR plants. In accordance with van Oorschot andLeeuwen (1984), we found a lower photosynthetic carbonassimilation rate in AR A. retroflexus, whereas the ratewas comparable in the AR and S biotypes of C. album,E. adenocaulon, E. canadensis, S. vulgaris and S. nigrum(Fig. 2). Differences in CO2 fixation, when expressedrelative to the total chlorophyll content of the leaves,may indicate an altered pigment stoichiometry in theleaves of AR plants.There are marked differences between the S and AR

biotypes in the allocation of the absorbed light energyto the PSII photochemistry and dissipative processes(Fig. 6).Modulated chlorophyll fluorescence measurementsmay furnish data of value for an assessment of the excit-ation energy fraction delivered to the open PSII centres(photochemistry) or thermally dissipated in comparisonsof wild andD1mutant biotypes of the same species. Assess-ments were carried out according to Demmig-Adams et al.(1996), assuming matching behaviour of PSI and PSII.Before the fluorescence measurements, plants were photo-inhibited at about 1500mmolm�2 s�1 PPFD, similar to theirgrowing conditions. The investigated species (wild types)showed characteristic differences in the case of theS biotypes, E. canadensis and S. vulgaris having the mostefficient PSII photochemistry, while S. nigrum mightsuffer worst from excessive excitation in nature. Therewas otherwise a quite surprising variability in the parti-tion of excessive excitation to thermal dissipation (D) andthe ultimate excess (X) that might generate reactiveoxygen species in the antennae and/or reduce molecularoxygen (Demmig-Adams et al. 1996). These assessmentsindicated that all of the studied AR biotypes exhibited

Table 2. In vitro violaxanthin de-epoxidase activity in the presenceof ascorbate in thylakoid membranes isolated from leaves ofatrazine-resistant (AR) and susceptible (S) biotypes of Chenopodiumalbum, Epilobium adenocaulon, Solanum nigrum and Erigeroncanadensis (V, violaxanthin; A, antheraxanthin; Z, zeaxanthin;n¼ 4–5).

Species Biotype pH (A1Z)/(V1A1Z)

Chenopodium album S 7.2 0.098� 0.0125.2 0.495� 0.040

AR 7.2 0.089� 0.0165.2 0.463� 0.034

Epilobium adenocaulon S 7.2 0.087� 0.0145.2 0.495� 0.035

AR 7.2 0.110� 0.0165.2 0.456� 0.042

Solanum nigrum S 7.2 0.030� 0.0055.2 0.376� 0.032

AR 7.2 0.050� 0.0045.2 0.412� 0.030

Erigeron canadensis S 7.2 0.068� 0.0045.2 0.485� 0.024

AR 7.2 0.078� 0.0055.2 0.496� 0.026

52 Physiol. Plant. 118, 2003

a more or less increased dissipative capacity. The mostimportant fact, however, is that all of the AR biotypesunequivocally displayed a diminished photochemicalefficiency.There seems to be a contradiction between this indica-

tion of a sometimes much higher assessed energy dissipa-tion and the lowered xanthophyll cycle activity (Fig. 3)and the higher light sensitivity indices of the AR plants(Fig. 5). One possible explanation for this is that ARplants might have alternative defence mechanisms, e.g.a markedly enhanced D1 protein turnover, which may beanother key element of photoprotection in PSII. Thequestion remains, however, of whether the assessmentof the allocation of the excessive excitation to thermaldissipation is fully adaptable to the AR biotype or not.The calculated light sensitivity index works well with thewild (S) biotypes and also indicates the higher photo-sensitivity of the AR plants. However, there is an anomal-ous region at low PPFDs (around 300mmolm�2 s�1)where some of the AR biotypes exhibit higher sensitivitythan at higher irradiances. It cannot be excluded, how-ever, that these D1 mutant plants may possess as yet

unknown special regulatory and/or dissipative processesthat do not fully match the preconditions of this assess-ment.The only well-marked strangeness in the pigment com-

position (Table 1) was the consistently lower chlorophylla/b ratio in the leaves of the AR plants relative to the Splants for all the investigated dicots. This may indicate ashift in the PSII/PSI ratio and/or (similarly as for shade-acclimated leaves, but in contrast not for a shaded habi-tat) a relatively larger light harvesting antenna in the ARbiotypes, and this latter might result in a higher excess ofexcitation energy in the photosynthetic apparatus ofthese D1 protein mutants. This ‘shade-type character’,together with a weak xanthophyll cycle performance,may well explain the high photosensitivity or excitationpressure (Fig. 5) in the AR biotypes of some species, evenat low PPFDs. At all PPFDs, floated leaf discs (Fig. 3)showed an unusually high rate of de-epoxidation ascompared with the intact leaves in vivo (Varadi et al.1994a, 1994b, Darko et al. 1996). This may be a conse-quence of the seriously reduced photosynthetic activity,due to the much lower diffusion velocities in the liquid

A

0

1

2

3

4

B

0

1

2

3

4

D

0

1

2

3

4

0.0

0.5

1.0

0.0

0.5

1.0

0.0

0.5

1.0

0.0

0.5

1.0

0.0

0.5

1.0

0.0

0.5

1.0

0.0

0.5

1.0

0.0

0.5

1.0

0.0

0.5

1.0

0.0

0.5

1.0

C

Non

-pho

toch

emic

al q

uenc

hing

(N

PQ

)

0

1

2

3

4

Phot

oche

mic

al q

uenc

hing

coe

ffic

ient

(q P)

Yie

ld o

f PS

II p

hoto

chem

istr

y (∆

F/F m

')

E

0

1

2

3

4

PPFD (µmol m–2 s–1)

0 500 100 1500 20000 500 100 1500 20000 500 100 1500 2000

Fig. 4. Light response of the non-photochemical fluorescence quenching(NPQ, left), the photochemical quenching(qP, middle) and the yield of PSIIphotochemistry (right) in the leaves ofatrazine-resistant (AR, open symbols) andsusceptible (S, closed symbols) biotypes ofChenopodium album (A), Epilobiumadenocaulon (B), Senecio vulgaris (C),Solanum nigrum (D) and Amaranthusretroflexus (E) (for similar data on Erigeroncanadensis, see Darko et al. 1996, 2000).The data are means of 4 replicates from 3independent experiments, and standarderrors are visible when larger than thesymbols.

Physiol. Plant. 118, 2003 53

phase and, as a consequence, the certainly higher excessof excitation in the leaf tissues under these conditions.However, there was an uncontradictable shortfall in thede-epoxidation in all the AR biotypes of the dicot weedsas compared with the wild biotypes, even under theseextreme circumstances, which may reflect differing avail-ability of V in the two biotypes in vivo.There were no significant and unequivocal differences

in xanthophyll cycle pool sizes between the S and ARplants in the studied dicot species (Table 1), so the differ-ences between the two biotypes in the xanthophyll cycleperformance and the NPQ process should be related tosome regulatory element, possibly to the trans-thylakoidDpH formation and the PsbS dose per PSII unit (Li et al.2002a). However, neither the psbS titre nor the extent ofthe light-induced DA535 absorbance change was investi-gated in these biotypes. Otherwise, even with the samePsbS levels in the two biotypes, an altered degree ofprotonation of PsbS due to restrained DpH formationunder high-light conditions in the AR plants cannot beexcluded. Point mutations in psbS affecting the numberor the position of the protonatable amino acid residuesin the PsbS on the luminal side, which might be criticalfor the proper functioning and stability (Li et al. 2002b),can also be hypothesized, although this has not beenstudied yet in these plant species.

The xanthophyll cycle activity (V de-epoxidation) hasbeen suggested to be limited by a temporary ascorbatedeficit in vivo (Muller-Moule et al. 2002). It has alsobeen shown that the regulation of xanthophyll cycle-dependent non-radiative energy dissipation in thepigment bed of PSII is modulated not only by lumenacidification, but also by ascorbate availability (Neubauerand Yamamoto 1994). The question arises of whether a

A

0.0

0.5

1.0

1.5

0.0

0.5

1.0

1.5

0.0

0.5

1.0

1.5

B

C

Phot

osen

sitiv

ity in

dex

or e

xcita

tion

pres

sure

(1

– q P)

/NPQ

D

E

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

0 500 1000 1500 2000 0 500 1000 1500 2000

F

Fig. 5. Light sensitivity indices ([1–qP]/NPQ) of the leaves ofatrazine-resistant (AR, open symbols) and susceptible (S, closedsymbols) biotypes of Chenopodium album (A), Epilobiumadenocaulon (B), Senecio vulgaris (C), Solanum nigrum (D),Erigeron canadensis (E) and Amaranthus retroflexus (F). The dataare calculated from the results presented in Fig. 4, and standarderrors are visible when larger than the symbols.

C. album 'S'

D

P X

D

P X

E. adenocaulon 'S'

D

P X

D

PX

E. canadensis 'S'

D

P

XD

P X

S. vulgaris 'S'

D

P

XD

PX

S. nigrum 'S'

D

P X

D

PX

A. retroflexus 'S'

D D

P PX X

A

B

C

D

E

F

C. album 'AR'

E. adenocaulon 'AR'

A. retroflexus 'AR'

E. canadensis 'AR'

S. vulgaris 'AR'

S. nigrum 'AR'

Fig. 6. Allocation pattern of excitation energy absorbed in the PSIIantennae to PSII photochemistry (P¼ (Fv

0/Fm0)*qP, black areas),

thermal dissipation (D¼ 1–Fv0/Fm

0, dark-grey areas) and theremaining excess excitation (X, light-grey areas) in the leaves ofatrazine-resistant (AR, right) and susceptible (S, left) biotypes ofChenopodium album (A), Epilobium adenocaulon (B), Seneciovulgaris (C), Solanum nigrum (D), Erigeron canadensis (E) andAmaranthus retroflexus (F). The data are calculated from theaverage values of the results presented in Fig. 4.

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 poisedin a more reduced state in the AR biotype as comparedwith the wild type, which can be explained by an alteredQA

–/QB equilibrium constant in the D1 mutant (Boweset al. 1980). The redox state of the plastoquinone poolmay affect the V availability (Siefermann and Yamamoto1975), i.e. a slower electron flow from the quinone accep-tors of PSII, resulting in a more oxidized state of the PQpool, may reduce the V availability. This may cause lowerlevels of Z relative to the whole xanthophyll cycle pool.The mechanism of this redox control on V availability isas yet unknown.The explanation of the lowered V de-epoxidation in

the AR biotypes may be hidden in the trans-thylakoidDpH-related xanthophyll cycle regulation processes. It isinferred that V de-epoxidation is controlled both at thelevel of enzyme activity and at the level of V availability(Pfundel et al. 1994). The two elements can be regulatedindependently. The activity of the VDE bound to theluminal side of the thylakoids probably depends on thepH of localized proton domains in the thylakoids, andthe V availability is controlled by the (delocalized) lumenpH, perhaps via the pH-dependent reoxidation of plasto-quinol by the Cyt b/f complex. It has been shown forE. canadensis (Darko et al. 2000), and confirmed forfurther dicot weeds, that the in vitro activity of VDEdoes not differ in the thylakoids of the S and AR bio-types near the optimum pH (5.2) and in the presence ofascorbate (Table 2). The enzyme regulation and activitiesin vivo, however, may be notably different from thoseobserved in vitro. Exactly the same in vivo light responseof the xanthophyll cycle was observed in intact attachedleaves of the S and AR biotypes of E. canadensis in thepresence of the strong artificial electron acceptor methyl-viologen (paraquat) (Darko et al. 2000, Varadi et al.2000) and confirmed for E. adenocaulon too (data notshown). These experimental findings indicate two import-ant things: (1) the maximum in vivo VDE activity andthe V availability should become similar in the biotypeswhen the natural mechanisms of lumen acidificationare disturbed by a strongly accelerated linear electrontransport and an extremely large trans-thylakoid DpHprobably arises, and (2) a relative ascorbate deficiencymight not be the cause of the weaker light response of Zformation in the AR biotype. Hence, a lower actual VDEactivity due to the lowered photosynthetic electron trans-port rate resulting in a reduced DpH formation appears tobe the possible explanation of the altered behaviour of thexanthophyll cycle and the NPQ process and the higherphotosensitivity in the AR dicots. A possible role of thePsbS protein must be investigated in relation to atrazineresistance.

Acknowledgements – This work was supported by HungarianResearch Fund (OTKA) grant No. T-035252.

References

Bowes JM, Crofts AR, Arntzen CJ (1980) Redox reactions on thereducing side of photosystem II in chloroplasts with altered herbi-cide binding properties. Arch Biochem Biophys 200: 303–308

Curwiel VB, Schansker G, de Vos OJ, van Rensen JJS (1993)Comparison of photosynthetic activities in triazine-resistantand susceptible biotypes of Chenopodium album. Z Naturforsch48c: 278–282

Darko E, Varadi Gy Dulai S, Lehoczki E (1996) Atrazine-resistantbiotypes of Conyza canadensis have altered fluorescence quench-ing and xanthophyll cycle pattern. Plant Physiol Biochem 34:843–852

Darko E, Varadi Gy Lemoine Y, Lehoczki E (2000) Defensivestrategies against high-light stress in wild and D1 protein mutantbiotypes of Erigeron canadensis. Aust J Plant Physiol 27: 325–333

Demmig B, Winter K, Kruger A, Czygan F-C (1987) Photo-inhibition and zeaxanthin formation in intact leaves. Plant Physiol84: 218–224

Demmig-Adams B, Adams WW III (1992) Photoprotection andother responses of plants to high light stress. Ann Rev PlantPhysiol Plant Mol Biol 43: 599–626

Demmig-Adams B, Adams WW, Barker DH, Logan BA,Bowling DR, Verhoeven AS (1996) Using chlorophyll fluorescenceto assess the fraction of absorbed light allocated to thermaldissipation of excess excitation. Physiol Plant 98: 253–264

Ducruet J, Lemoine Y (1985) Increased heat sensitivity of thephotosynthetic apparatus in triazine-resistant biotypes from dif-ferent plant species. Plant Cell Physiol 26: 419–429

Ducruet J, Ort DR (1988) Enhanced susceptibility of photosynth-esis to high leaf temperature in triazine-resistant Solanumnigrum L. evidence for photosystem II, D1 protein site of action.Plant Sci 56: 39–48

Dulai S, Molnar I, Lehoczki E (1998) Effect of growth temperaturesof 5 and 25�C on long-term responses of photosystem II to heatstress in atrazine-resistant and susceptible biotypes of Erigeroncanadensis. Aust J Plant Physiol 25: 145–153

Eskling M, Arvidsson P-O, Akerlund H-E (1997) The xanthophyllcycle, its regulation and components. Physiol Plant 100: 806–816

Genty B, Briantais JM, Baker NR (1989) The relationship betweenthe quantum yield of non-photochemical quenching of chloro-phyll fluorescence and the rate of photosystem 2 photochemistryin leaves. Biochim Biophys Acta 990: 87–92

Gilmore AM (1997) Mechanistic aspects of xanthophyll cycle-dependent photoprotection in higher plant chloroplasts andleaves. Physiol Plant 99: 197–209

Gilmore AM, Shinkarev VP, Hazlett TL, Govindjee (1998)Quantitative analysis of the effects of intrathylakoid pH andxanthophyll cycle pigments on chlorophyll a fluorescencelifetime distributions and intensity in thylakoids. Biochemistry37: 13582–13593

Gressel J (1985) Herbicide tolerance and resistance: alteration ofsite of activity. In: Duke, SO (ed) Weed Physiology. II CRCPress, Boca Raton, Florida, pp 159–189

Hager A, Holocher K (1994) Localization of the xanthophyll-cycleenzyme violaxanthin de-epoxidase within the thylakoid lumenand abolition of its mobility by a (light-dependent) pH decrease.Planta 192: 581–589

Hart JJ, Stemler A (1990) Similar photosynthetic performance inlow light grown isonuclear triazine-resistant and – susceptiblebiotypes of Brassica napus L. Plant Physiol 94: 1295–1300

Havaux M (1989) Comparison of atrazine-resistant and –susceptible biotypes of Senecio vulgaris L. effect of high andlow temperatures on the in vivo photosynthetic electron transferin intact leaves. J Exp Bot 40: 849–854

Hirschberg J, Blecker A, Kyle DJ, McIntosh L, Arntzen CJ (1984)The molecular basis of triazine-herbicide resistance in higherplant chloroplasts. Z Naturforsch 39c: 412–420

Horton P, Ruban AV, Walters RG (1996) Regulation of lightharvesting in green plants. Ann Rev Plant Physiol Plant MolBiol 47: 655–684

Jansen MAK, Pfister K (1990) Conserved kinetics at thereducing side of reaction-center II in photosynthetic organisms;changed kinetics in triazine-resistant weeds. Z Naturforsch 45c:441–445

Physiol. Plant. 118, 2003 55

Li X-P, Bjorkman O, Shih C, Grossman AG, Rosenquist Jansson S,Niyogi KK (2000) A pigment-binding protein essential for regul-ation of photosynthetic light harvesting. Nature 403: 391–395

Li X-P, Gilmore AM, Niyogi KK (2002a) Molecular and globaltime-resolved analysis of a psbS gene dosage effect on pH- andxanthophyll cycle-dependent nonphotochemical quenching inphotosystem II. J Biol Chem 277: 33590–33597

Li X-P, Phippard A, Pasari J, Niyogi KK (2002b) Structure-function analysis of photosystem II subunit S (PsbS) in vivo.Funct Plant Biol 29: 1131–1139

Lichtenthaler HK (1987) Chlorophylls and Carotenoids: Pigmentsof Photosynthetic Biomembranes. In: Packer, L, Douce, R (eds)Methods in Enzymology, Vol. 148. Academic Press, New York,NY, pp 350–382

McCloskey WB, Holt JS (1991) Effect of growth temperature onbiomass production of nearly isonuclear triazine-resistant and –susceptible common groundsel (Senecio vulgaris L.). Plant CellEnviron 14: 699–705

Muller P, Li X-P, Niyogi KK (2001) Non-photochemical quenching.A response to excess light energy. Plant Physiol 125: 1558–1566

Muller-Moule P, Conklin PL, Niyogi K (2002) Ascorbate deficiencycan limit violaxanthin de-epoxidase activity in vivo. PlantPhysiol 128: 970–977

Neubauer C, Yamamoto H (1994) Membrane barriers andMehler-peroxidase reaction limit the ascorbate available forviolaxanthin de-epoxidase activity in intact chloroplasts.Photosynth Res 39: 137–147

van Oorschot JLP, Leeuwen PH (1984) Comparison of the photo-synthetic capacity between intact leaves of triazine-resistant and –susceptible biotypes of six weed species. ZNaturforsch 39c: 440–442

Osmond CB (1994) What is photoinhibition? Some insights fromcomparisons of shade and sun plants. In: Baker, NR, Bowyer, JR(eds) Photoinhibition of Photosynthesis: From MolecularMechanisms to Field, Environmental Plants Biology. BIOS,Oxford, pp 1–24

Pfister K, Arntzen CJ (1979) The mode of action of photosystemII-specific inhibitor in herbicide-resistant weed biotypes.Z Naturforsch 34c: 996–1009

Pfundel E, Bilger W (1994) Regulation and possible function ofthe violaxanthin cycle. Photosynth Res 42: 89–109

Pfundel E, Renganathan M, Gilmore AM, Yamamoto HY, DilleyRA (1994) Intrathylakoid pH in isolated pea chloroplasts asprobed by violaxanthin de-epoxidation. Plant Physiol 106:1647–1658

Schreiber U, Schliwa U, Bilger W (1986) Continuous recording ofphotochemical and non-photochemical chlorophyll fluorescencequenching with a new type of modulation fluorometer.Photosynth Res 10: 51–62

Shen Y-K, Chow WS, Park Y-I, Anderson JM (1996) Photo-inactivation of photosystem II by cumulative exposure to shortlight pulses during the induction period of photosynthesis.Photosynth Res 47: 51–59

Siefermann D, Yamamoto HY (1975) Light-induced de-epoxidationof violaxanthin in lettuce chloroplasts. IV. The effects of electron-transport conditions on violaxanthin availability. BiochimBiophys Acta 387: 149–158

Sundby C, Chow WS, Anderson JM (1993) Effects on photosystemII, photoinhibition, and plant performance of the spontaneousmutation of serine-264 in the photosystem II reaction center D1protein in triazine-resistant Brassica napus L. Plant Physiol 103:105–113

Varadi Gy, Botos-Balo B, Polos E (1992) Xanthophyll cycle ingrapevine leaves – diurnal and seasonal patterns. Proceedingsof the IVth International Symp on Grapevine Physiology, 11–15May. San Michele All’adige, Torino, pp 521–526

Varadi Gy, Darko E, Lehoczki E (2000) Changes in the xanthophyllcycle and fluorescence quenching indicate light-dependent earlyevents in the action of paraquat and the mechanism of resistanceto paraquat in Erigeron canadensis. Plant Physiol 123:1459–1469

Varadi Gy, Darko E, Polos E, Szigeti Z, Lehoczki E (1994a)Xanthophyll cycle patterns and in vivo photoinhibition in herbi-cide-resistant biotypes of Conyza canadensis. J Plant Physiol144: 669–674

Varadi Gy, Darko E, Polos E, Szigeti Z, Lehoczki E (1994b) Xantho-phyll cycle pattern and in vivo photoinhibition in atrazine-resistant biotypes of Conyza canadensis and Chenopodiumalbum. Proceedings of the 9th Congress of the Federation ofEuropean Societies Plant Physiology, Brno (Czech Republic),3–8 July. Biol Plant, 36 (Suppl.): S168

Warwick SI, Black L (1981) The relative competitiveness of atrazinesusceptible and resistant populations of Chenopodium album andC. strictum. Can J Bot 59: 689–693

Yamamoto HY, Bassi R (1996) Carotenoids: Localization andfunction. In: Ort, RR, Yocum, CF (eds) Oxygenic Photo-synthesis: the Light Reactions. Kluwer Academic Publishers,Dordrecht, pp 539–563

Edited by A. J. Stemler

56 Physiol. Plant. 118, 2003