relationship between the degree of carotenoid depletion and function of the photosynthetic apparatus

Journal of Photochemistry and Photobiology B: Biology 96 (2009) 49–56

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier .com/locate / jphotobiol

Relationship between the degree of carotenoid depletion and functionof the photosynthetic apparatus

Kolyo Dankov a, Mira Busheva a, Detelin Stefanov b, Emilia L. Apostolova a,*

a Institute of Biophysics, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113, Bulgariab Institute of Plant Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113, Bulgaria

a r t i c l e i n f o

Article history:Received 12 December 2008Received in revised form 25 March 2009Accepted 6 April 2009Available online 14 April 2009

Keywords:CarotenoidsChlorophyll fluorescenceFluridonePhotosynthetic apparatus

1011-1344/$ - see front matter � 2009 Elsevier B.V. Adoi:10.1016/j.jphotobiol.2009.04.004

* Corresponding author. Tel.: +359 2 979 26 21; faxE-mail address: emya@obzor.bio21.bas.bg (E.L. Ap

a b s t r a c t

Fluridone, an inhibitor of the carotenoid biosynthesis, was used to study the relationship between thedegree of carotenoid depletion and the function of the photosynthetic apparatus. The data reveal that,at a small reduction of the carotenoid content (25% decrease of the total carotenoids), the PSII and PSI(oxidation of P700 by far-red light) photochemistry is not influenced, while the oxygen evolution isstrongly inhibited. Further reduction of the total carotenoid content (more than 40%) leads to decreaseof the chlorophyll content and inhibition of the functions of both photosystems as the effect on the pho-tosynthetic oxygen evolution and primary photochemistry is stronger than the effect on P700 oxidation.The analysis of the oxygen production under continuous illumination and flash oxygen yields suggeststhat the inhibition of the oxygen evolution is caused mainly by the damage of PSIIa centers.

� 2009 Elsevier B.V. All rights reserved.

1. Introduction

Carotenoids located in the antenna and core complexes of bothphotosystems, are important for the functions and stability of pig-ment–protein complexes [1–3]. They are light-harvesting acces-sory pigments and have a special role in the thermal dissipationof excess light energy [4–5]. The most important function ofcarotenoids is the photoprotection of the photosynthetic apparatusby quenching triplet chlorophyll, singlet oxygen and other reactivespecies [6]. It was demonstrated that there are two b-carotenemolecules in the core complex of photosystem II (PSII) which pro-tect chlorophyll P680 from photodamage [7–10] and this could berelated to protection against degradation of the D1 subunits of PSII[11]. Moreover, it has been ascertained that carotenoids can alsooperate in thylakoid membranes as a stabilizer of the lipid phase[12].

Carotenoids are structural components of light-harvesting chlo-rophyll a/b protein complexes of PSII (LHCII) and photosystem I(PSI). They are involved in the stabilization of the LHCII trimersas well as in assembly of LHCII monomers [13–15].

One approach to elucidate the role of carotenoids in vivo isthrough the use of bleaching herbicides which inhibit carotenoidbiosynthesis. Certain classes of herbicides are interfering withcarotenoid biosynthesis leading to pigment destruction and ableached plant phenotype. Considerable reduction in the pigment

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: +359 2 971 24 93.ostolova).

content finally results in plant death because of missing photopro-tection [16].

One of the important target sites for bleaching herbicides is theenzyme phytoene desaturase catalysing the desaturation of phyto-ene into phytofluene in the carotenoid biosynthesis pathway. Nor-flurazon or fluridone can inhibit this enzymatic reaction [17]. DiBaccio et al. [18] have shown that the treatment of etioplasts withbleaching herbicide (amitrole and norflurazon) leads to changes inthe lipid composition and fatty acid unsaturation of the individualpolar lipids, as well as to a decrease of the lipid to protein ratio.Using bleaching herbicides, norflurazon and fluridone, Trebst andDepka [19] have concluded that b-carotene is essential for theassembly of D1 protein into functional PSII. They proposed thatbleaching of b-carotene in the reaction center of PSII by high lightdestabilizes the structure and triggers the degradation of the D1protein.

While the protective role of carotenoids against oxidative dam-age is largely documented, the effect of different degree of caroten-oid depletion on the function of the photosynthetic apparatus isstill unclear. The present work provides new evidence of the influ-ence of the degree of carotenoid depletion on the function of peathylakoid membranes. The analysis of the data from low and roomtemperature chlorophyll fluorescence, oxygen evolution usingpolarographic oxygen rate electrode and P700 redox state mea-surements show that the small reduction of the total carotenoidcontent (25% decrease) influences mainly the oxygen evolvingactivity of the photosynthetic apparatus, while the photochemistryof both photosystems is not affected. Further decrease in thecarotenoids (more than 40%) leads to inhibition of both

50 K. Dankov et al. / Journal of Photochemistry and Photobiology B: Biology 96 (2009) 49–56

photosystems as the effect on the oxygen evolution and primaryphotochemistry is stronger than the effect on P700 oxidation.

2. Material and methods

2.1. Plant material and preparation of thylakoid membranes

The plants from Pisum sativum L. cv. Ran were grown hydropon-ically under controlled conditions with 16 h light/8 h dark periodin the presence of different concentrations of fluridone (1-methyl-3-phenyl-5-(3-trifluoromethylphenyl)-4-(1H)-pyridone).Thylakoid membranes were isolated from 14-day-old pea plants,as described in [20] and suspended in a medium containing:40 mM HEPES (pH 7.6), 10 mM NaCl, 5 mM MgCl2 and 400 mMsucrose.

2.2. Pigment analysis

The pigments were extracted from pea leaves with 80% acetone.Chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids (Car)were determined spectrophotometrically according to Lichtenthal-er [21].

2.3. Room temperature chlorophyll a fluorescence

Modulated Chl fluorescence was measured on leaf discs by aPAM fluorometer (H. Walz, Effeltrich, Germany, model PAM 101–103). The leaves were dark adapted for 15 min. The F0 level wasmeasured at instrument frequency of 1.6 kHz and measuring beamset at 0.020 lmol m�2 s�1 PFD. For evaluation of maximum fluores-cence level, saturating flashes of 3000 lmol m�2 s�1 PFD withduration of 0.8 s were provided by Schott lamp KL 1500 (SchottGlaswerke, Mainz, Germany). The time interval between two con-secutive flashes was 20 s. The saturating flash gives Fm in dark-adapted state (DAS) and F 0m – in light-adapted state (LAS). The ac-tinic light illumination (250 lmol m�2 s�1 PFD) was provided bysecond Schott lamp KL 1500 for the induction of photosynthesis.The steady state level F 0s was estimated after 4 min illuminationwith actinic light. The minimum chlorophyll fluorescence in LAS,F 00, was calculated by the expression of Oxboroux and Baker,F 00 ¼ F0=ðFv=Fm þ F0=F 0mÞ [22]. Using the fluorescence levels: Fm,F0, F 0s; F

0m and F 00 of DAS and LAS the following parameters were cal-

culated: maximum quantum yield for primary photochemistry inDAS, UPo = (Fm � F0)/Fm = Fv/Fm [23], which is a function of the ratiobetween the rate constants of photochemical (kP) and non-photo-chemical processes (kN) of PSII antenna de-excitation, UPo = kP/(kN + kP) [24]; the photochemical quenching coefficient in LAS,q0P ¼ ðF

0m � F 0sÞðF

0m � F 00Þ [25]; ratio of the rate constants for non-

photochemical quenching events in LAS versus DAS,k0N=kN ¼ Fm=F 0m [26]; the maximum quantum yield for primaryphotochemistry in LAS, U0Po ¼ F 0v=F 0m ¼ k0P=ðk

0N þ k0PÞ [27], which

gives the efficiency of excitation capture by open PSII reaction cen-ters; the actual quantum yield for primary photochemistry in LAS,U0Ps ¼ ðF

0m � F 0sÞ=F 0m [28], which is equal to the product of

q0P and F 0v=F 0m [29]; relative variable fluorescence in LAS (degreeof PSII reaction center closure), V 0s ¼ 1� q0P ¼ ðF

0s � F 00Þ=ðF

0m � F 00Þ

[30].

2.4. Low temperature chlorophyll a fluorescence

Low temperature (77 K) chlorophyll fluorescence measure-ments of isolated thylakoid membranes from control and fluridonetreated plants were performed as in [31]. Thylakoid membraneswere suspended in a medium containing: 40 mM HEPES (pH 7.6),10 mM NaCl, 5 mM MgCl2 and 400 mM sucrose. The chlorophyllconcentration was 10 lg chl/ml. Chlorophyll fluorescence was ex-

cited either at 436 nm (Chl a), at 472 nm (Chl b) or at 515 nm(Car). For correct measurements of fluorescence yield the 0.5 lMfluorescein is added to the suspension as an internal standard. Atthis concentration the fluorescein did not interfere with the fluo-rescence emission [32]. The emission spectra were normalized atthe maximum fluorescence intensity of the standard (506 nm).

2.5. Oxygen evolution measurements

Oxygen flash yields and initial oxygen burst of isolated thyla-koid membranes were measured by a home-built polarographicoxygen rate electrode described in [33]. Thylakoid membraneswere suspended in a medium containing: 40 mM HEPES (pH 7.6),10 mM NaCl, 5 mM MgCl2 and 400 mM sucrose. The chlorophyllconcentration was 150 lg chl/ml. The measurements were per-formed as in [34]. The induction curves after oxygen burst exhibitbiphasic exponential decay. The deconvolution of the oxygen burstdecay was performed as in [31]. Initial S0 and S1 state distribution,misses (a) and double hits (b) were determined by the fitting of thetheoretically calculated yields according to the model of Kok et al.[35] with the experimentally obtained oxygen flash yields usingthe least square deviations procedure.

2.6. P700 redox state measurements

The redox state of P700 was investigated on leaf discs with adual wavelength (810/860 nm) unit (Walz ED 700DW-E) attachedto a PAM101E main control unit in the reflectance mode as in [36].

2.7. Statistical analysis

The results are mean values from 3 to 5 independent experi-ments. The statistical differences between the means were deter-mined using a two-tailed paired Student’s t-test.

Values of P < 0.05 were considered as significantly different be-tween the fluridone treated samples and the untreated sample.

3. Results

3.1. Fluridone-induced changes in the content of photosyntheticpigments

Analysis of the pigment composition after fluridone treatmentof pea plants indicates that carotenoid biosynthesis inhibitorcauses a decrease in both the Car and total chlorophyll content,depending on the herbicide concentration. The decrease of theChl and Car content is at different extent (Table 1). At higher fluri-done concentration (10�6 M) the total Chl content decreases with59%, while the Car content with 67%, which leads to increase ofthe Chl/Car ratio. On the other hand the content of Chl a decreasesmore obviously than Chl b, i.e. the Chl a/b ratio also diminished. Thechanges in the ratio Chl a/b are bigger than in the ratio Chl/Car athigher concentration of the herbicide (10�6 M). The lower fluridoneconcentration (10�8 M) has a slight effect on chlorophyll content,while the carotenoids decrease by 25% (Table 1). Depending onthe degree of Car depletion we introduced the following abbrevia-tions: 25CarD (the plants treated with 10�8 M fluridone, which leadto 25% Car depletion), 40CarD (the plants treated with 10�7 M fluri-done, which lead to 40% Car depletion) and 65CarD (the plants trea-ted with 10�6 M fluridone, which lead to 65% Car depletion).

3.2. Fluridone-induced changes in 77 K fluorescence emission spectraof thylakoid membrane

We demonstrate that under fluridone treatment there is dimin-ishing of Car and Chl content along with changes in Chl a/b ratio

Table 1Pigment content of leaves from plants treated with different fluridone concentration. Mean values ± SE are from five independent experiments.

Concentration (M) Chl a (mg/g FW) Chl b (mg/g FW) Car (mg/g FW) Chl a/b Chl/Car

0 2.68 ± 0.10 (100%) 1.30 ± 0.06 (100%) 0.82 ± 0.04 (100%) 2.06 ± 0.04 4.85

10�8 2.30 ± 0.08* (86%) 1.21 ± 0.04 (93%) 0.62 ± 0.05* (76%) 1.90 ± 0.05* 5.66

10�7 1.61 ± 0.09*** (60%) 0.95 ± 0.07** (73%) 0.47 ± 0.04*** (58%) 1.69 ± 0.09** 5.45

10�6 0.95 ± 0.04*** (35%) 0.70 ± 0.03*** (54%) 0.27 ± 0.02*** (33%) 1.35 ± 0.08*** 6.11

The asterisks mark significant differences between the untreated sample and the fluridone treated samples.* P < 0.05.** P < 0.01.*** P < 0.001.

K. Dankov et al. / Journal of Photochemistry and Photobiology B: Biology 96 (2009) 49–56 51

(Table 1). To elucidate the effect of decreased pigment content andcorresponding changes on the chlorophyll fluorescence propertiesof the photosynthetic apparatus we measured the 77 K fluores-cence emission spectra of thylakoid membranes isolated frompea plants treated with 10�8 and 10�7 M fluridone. The steady-state fluorescence emission spectra (kexc 436 nm) of the thylakoidmembranes are shown in Fig. 1. The line shapes have two clearlyexpressed maxima at 686 nm (F1) and 737 nm (F3) and a shoulderat 695 nm (F2). The emission bands at 686 nm and 695 nm are re-lated to PSII, and the one around 735 nm to PSI [37–38]. The calcu-lated overall fluorescence yield of thylakoid membranes afternormalization of the spectra to fluorescein (see Section 2) revealeda decline almost to 50% with increasing of fluridone concentration(9% for 10�8 M and 47% for 10�7 M). Analysis of the fluorescenceemission spectra shows that Car depletion influences the intensityof the main bands of chlorophyll–protein complexes in the thyla-koid membranes (Fig. 1). The calculated ratio between the intensi-ties of main bands, when different excitation wavelengths are used(436 nm for Chl a, 472 nm for Chl b or 515 nm for Car) is shown inTable 2. The PSI/PSII fluorescence ratio (F3/F1) decreases slightly(14–19%) at the lower fluridone concentration (10�8 M) and by30–34% at the higher concentration (10�7 M) for all wavelengthsof excitation. The herbicide treatment (10�7 M fluridone) leads tosmall (10–14%) increase of the F2/F1 ratio only for excitation at

Fig. 1. Effect of carotenoid depletion on 77 K fluorescence emission spectra (kexc

436 nm) of isolated pea thylakoid membranes from plants treated with differentfluridone concentrations: untreated (solid line); treated with 10�8 M fluridone(doted line); treated with 10�7 M fluridone (dash-doted line). The thylakoidmembranes were suspended in a medium containing: 40 mM HEPES (pH 7.6),10 mM NaCl, 5 mM MgCl2 and 400 mM sucrose. The chlorophyll concentration was10 lg chl/ml. The spectra were normalized at maximum fluorescence intensity (at507 nm) of fluorescein. The presented spectra are representative from threeindependent experiments.

472 nm and 515 nm (Table 2). Moreover at this concentration theemission band at 737 nm is blue shifted to 734 nm compared tothe control for all excitation wavelength used (Fig. 1). It pointsout to a subsequent structural changes caused by fluridone treat-ment in the vicinity of PSI complex.

3.3. Influence of fluridone on the parameters of the room temperaturechlorophyll fluorescence

In order to characterize the physiological properties of plantsafter treatment with different fluridone concentrations we usedroom temperature chlorophyll a fluorescence. Table 3 shows thefluorescence parameters of control and fluridone treated plants.The maximum quantum yield of primary photochemistry in DAS(UPo = Fv/Fm) and LAS ðU0Po ¼ F 0v=F 0mÞ as well as the ratio of non-pho-tochemical quenching events in LAS versus DAS ðk0N=kN ¼ Fm=F 0mÞare influenced in the plants treated with concentration of fluridonehigher than 10�7 M (i.e. more than 40CarD), while the actual quan-tum yield for primary photochemistry in LAS ðU0Ps ¼ ðF

0m � F 0sÞ=F 0mÞ

is influenced even at the lowest studied herbicide concentration(10�8 M, i.e. 25CarD). UPo and U0Po for the plants treated with10�8 M fluridone are similar to the control plants, which suggestthat the primary photochemistry was not influenced at 25CarD.Having in mind that these parameters (UPo = kP/(kN + kP) andU0Po ¼ k0P=ðk

0N þ k0PÞ) are function of the rate constants of photo-

chemical ðkP; k0PÞ and non-photochemical ðkN; k

0NÞ de-excitation

processes in PSII antenna [24], it could be supposed that these pro-cesses are not influenced at 25CarD. A decline of U0Ps, which couldbe expressed as a product of q0P and F 0v=F 0mðU

0Ps ¼ q0P �U

0Po ¼

q0P � F0v=F 0mÞ is significant (about 90%) for the plants treated with

10�6 M fluridone. The drop of U0Ps at more than 40CarD is accompa-nied by a reduction of both photochemical quenching in LASðq0PÞ and F 0v=F 0m (Table 3), while at 25CarD the changes in U0Ps

mainly come from a decrease of the q0P.An important factor in determining the rate of PSII photochem-

istry is the redox state of QA, i.e. the fraction of PSII reaction centersthat are open and capable of photochemistry. The fraction of closedreaction centers in LAS is a function of the changes in the ratio ofreduced QA to total QA ðV 0s ¼ Q�A=QA totalÞ. The parameter V 0s rangesfrom 0 to 1, i.e. when V 0s ¼ 0 all the centers are open, while atV 0s ¼ 1 all the centers are closed. Data on Table 3 clearly show a de-crease of the fraction of open reaction centers with increase of thefluridone concentration ðV 0s increased).

3.4. Effect of fluridone on the photosynthetic oxygen evolution

Oxygen induction curves under continuous illumination of con-trol and fluridone treated plants are presented in Fig. 2. The induc-tion curves after oxygen burst exhibit biphasic exponential decay,which could originate from the existence of two different mecha-nisms and/or types of PSII centers for oxygen production [39].The parameters, used for the estimation of the effect of herbicide

Table 2Low temperature (77 K) fluorescence ratios of isolated thylakoid membranes from untreated and fluridone treated pea plants. F1 is the fluorescence emitted at 685. F2 is thefluorescence emitted at 695 nm and F3 nm is the fluorescence emitted at 737 nm for untreated and 10�8 M fluridone treated plants and emitted at 734 nm for 10�7 M fluridonetreatment. Excitation of Chl a (kexc 436 nm), Chl b (kexc 472 nm) and Car (kexc 515 nm). Mean values ± SE are from five independent experiments.

Concentration (M) kexc (nm) F3/F1 F2/F1

0 436 1.44 ± 0.09 0.73 ± 0.04472 1.25 ± 0.08 0.87 ± 0.02515 1.74 ± 0.09 0.87 ± 0.04

10�8 436 1.24 ± 0.08 0.71 ± 0.02472 1.07 ± 0.07 0.91 ± 0.04515 1.41 ± 0.08* 0.89 ± 0.04

10�7 436 1.01 ± 0.07** 0.75 ± 0.02472 0.88 ± 0.06** 0.99 ± 0.04*

515 1.15 ± 0.05*** 0.96 ± 0.02*

The asterisks mark significant differences between the fluridone treated simples and the untreated samples.* P < 0.05.** P < 0.01.*** P < 0.001.

Table 3Parameters from room temperature chlorophyll fluorescence of plants treated with different fluridone concentrations: maximum quantum yield for primary photochemistry inDAS, UPo = kP/(kN + kP); maximum quantum yield for primary photochemistry in LAS, U0Po ¼ k0P=ðk

0N þ k0PÞ; actual quantum yield for primary photochemistry in LAS, U0Ps;

photochemical quenching coefficient in LAS, q0P; relative variable fluorescence in LAS, V 0s; ratio of the rate constants for non-photochemical quenching events in LAS versus DAS,k0N=kN. For details see Section 2. Values are means ± SE.

Parameters Fluridone concentration (M)

0 10�8 10�7 10�6

UPo = Fv/Fm 0.81 ± 0.04 0.79 ± 0.04 0.51 ± 0.03*** 0.40 ± 0.02***

U0Po ¼ F 0v=F 0m 0.68 ± 0.03 0.64 ± 0.03 0.44 ± 0.02*** 0.13 ± 0.01***

U0Ps ¼ ðF0m � F 0sÞ=F 0m 0.50 ± 0.02 0.35 ± 0.02** 0.19 ± 0.02*** 0.05 ± 0.01***

q0P ¼ ðF0m � F 0sÞ=F0v 0.73 ± 0.04 0.55 ± 0.04** 0.43 ± 0.03*** 0.38 ± 0.03***

V 0s ¼ ðF0s � F00Þ=F0v 0.27 ± 0.02 0.45 ± 0.02** 0.57 ± 0.03*** 0.62 ± 0.03***

k0N=kN ¼ Fm=F 0m 3.03 ± 0.08 2.94 ± 0.08 2.37 ± 0.06*** 2.33 ± 0.05***

The asterisks mark significant differences between the fluridone treated samples and the untreated samples.** P < 0.01.*** P < 0.001.

52 K. Dankov et al. / Journal of Photochemistry and Photobiology B: Biology 96 (2009) 49–56

treatment on the oxygen evolution under continuous illuminationare: the amplitude of the oxygen burst (A, correlates with theamount of the functionally active PSII centers), the ratio betweenthe amplitudes of the fast (A1) and slow (A2) components (with rateconstants k1 and k2) of the oxygen burst decay (Fig. 2A and B). Theratio A1/A2 is used to estimate the proportion of the functionallyactive PSIIa and PSIIb centres [39,34]. The parameters obtainedfrom the analysis of the induction curves are shown in Table 4.

The oxygen burst under continuous illumination (A) decreases,depending on the fluridone concentration or degree of Car deple-tion (36% at 25CarD and 63% at 40CarD) (Table 4). The decreaseof the oxygen burst is accompanied by a diminishing of the ratioA1/A2, which is attributed to an inhibition of functionally activePSIIa centers evolving oxygen through a non-cooperative mecha-nism. The two rate constants k1 and k2 (Table 4) are smaller in thy-lakoid membranes from fluridone treated plants in comparison tountreated, which could be a consequence of modification in boththe PSIIa and PSIIb centers and/or altered interaction between QB

and PQ pool molecules.Oscillation patterns of the oxygen flash yields of thylakoid

membranes from treated and untreated plants are shown inFig. 3. The fluridone treatment strongly influences the flash oxygenevolution, which is produced by the PSIIa centers (Fig. 3, Table 4).The amplitude of the oxygen flash yields after the third flash (Y3)gradually decreases with the rise of the fluridone concentration.The effect on the flash oxygen evolution is stronger than on theoxygen burst after continuous illumination (Table 4). Even at25CarD the inhibition is about 70% as compared to the controlsamples. The inhibition of this process is related to an increase inthe S0 state population in the dark-adapted state, as well as in

the misses (a) and double hits (b) (Table 4). The observed varia-tions in the parameters of the flash oxygen evolution after fluri-done treatment could be a result from modification in thestructure of the PSII complex induced by decrease in the Carcontent.

3.5. Effect of fluridone on oxidation of P700 by far-red light

The effect of fluridone on PSI reactions was studied using theabsorbance changes around 830 nm, which are caused by the oxi-dation of P700 by far-red light and the subsequent reduction in thedark. Fig. 4 shows original traces of far-red light induced P700 oxi-dation in leaves from control and fluridone treated plants. A smalldecrease of the Car content (after treatment with 10�8 M fluridone)does not influence the P700 oxidation. The effectiveness of far-redlight in maintaining P700 in an oxidized state drops with furtherincrease of fluridone concentration. The post-illumination kineticsof P700+ re-reduction in the control leaves are well fitted by thesum of two negative exponents with rate constants around1.0 s�1 and 0.1 s�1. The analysis of the curves after treatment withfluridone concentrations higher than 10�7 M (more than 40CarD)shows lowering of the slow component (Is) of P700+ reduction(Fig. 5).

4. Discussion

Carotenoid biosynthesis is one of the major targets for bleach-ing herbicides. Treatment with fluridone leads to the inhibitionof phytoene desaturase and a decrease of the amount of carote-noids in the thylakoid membranes [17]. Our results reveal that

Fig. 2. Time course of the initial oxygen burst (induction curve) at continuousirradiation (450 lmol photon m�2 s�1) of pea thylakoid membranes isolated fromplants treated with different fluridone concentrations. (A) deconvolution of theoxygen burst decay of thylakoid membranes from untreated plant; (B) deconvo-lution of the oxygen burst decay of thylakoid membranes from plant treated with10�7 M; line 1 – fast component (A1) and line 2 – slow component (A2); (C) –comparison of the induction curves of thylakoid membranes from untreated (a) andplants treated with 10�7 M fluridone (b) (for details see Section 2). Thylakoidmembranes were suspended in a medium containing: 40 mM HEPES (pH 7.6),10 mM NaCl, 5 mM MgCl2 and 400 mM sucrose. The chlorophyll concentration was150 lg chl/ml. Time constant of the electrode is less than 2 ms. Polarographsensitivity is 1.5 V/lA.

Table 4Kinetic parameters of the oxygen flash yields and the initial oxygen burst undercontinuous irradiation of thylakoid membranes from plants, treated with differentfluridone concentrations. A and Y3 are represented as percentage of the values forthylakoid membranes from untreated plants. A1 and A2 are the amplitudes and k1 andk2 are the rate constants of the fast and slow components of the oxygen burst decay,respectively. Initial dark S0 and S1 states distribution in percentage of the total S states(S1 = 100 � S0), values of misses (a) and double hits (b) according to the Kok’s model.Average data are from five independent experiments.

Parameter Concentration (M)

0 10�8 10�7

A (%) 100 64 37A1/A2 2.45 1.97 1.54k1 (s�1) 4.55 4.12 3.87k2 (s�1) 0.61 0.58 0.54Y3 (%) 100 29 15S0 (%) 24 25 27a (%) 25 27 29b (%) 3.7 4.2 4.5

Fig. 3. Oscillation patterns of the oxygen flash yields in dark-adapted thylakoidmembranes from plants: (1) untreated, (2) treated with 10�8 M and (3) treated with10�7 M fluridone. Thylakoid membranes were suspended in a medium containing:40 mM HEPES (pH 7.6), 10 mM NaCl, 5 mM MgCl2 and 400 mM sucrose. Thechlorophyll concentration was 150 lg chl/ml. Time constant of the electrode is lessthan 2 ms. Polarograph sensitivity is 3 V/lA.

Fig. 4. Original traces of P700 photo-oxidation by far-red light and subsequentreduction of P700+ (decay signal) in leaves from control and plants treated withdifferent fluridone concentrations: (1) – untreated; (2) – treated with 10�8 Mfluridone; (3) – treated with 10�7 M fluridone and (4) – treated with 10�6 Mfluridone. Each trace is the average from five separate leaf discs.

K. Dankov et al. / Journal of Photochemistry and Photobiology B: Biology 96 (2009) 49–56 53

with growing of the herbicide concentration the carotenoid con-tent decreases as well as the amount of the chlorophylls (Table1). Kim et al. [16] have shown similar changes of the Car and totalchlorophyll after herbicide treatment. It was suggested that the de-crease of the chlorophylls could be a result of a carotenoid defi-ciency-induced photooxidation of the chlorophylls. In addition,we show that the degradation of Chl b is smaller than that of Chla (Table 1). Such a decrease in the chlorophyll content and Chl a/b ratio could be attributed to damage of the chlorophyll a-bindingproteins. This fact is in agreement with the observation that coreantenna complexes of PSII are more sensitive to illumination thanthe peripheral complexes [40]. The degradation of these pigment–protein complexes could be due to the degradation of their newly

Fig. 5. Semi-logarithmic plot showing the changes of the amplitudes of slow (N),fast (d) and total (j) component of P700+ re-reduction signal in leaves from controland plants treated with different fluridone concentration.

54 K. Dankov et al. / Journal of Photochemistry and Photobiology B: Biology 96 (2009) 49–56

synthesized molecules, as a consequence of chlorophyll photooxi-dation [41], which influences pigment–protein interaction andapoprotein stabilization [42].

It has been shown that the changes in the pigment compositionafter treatment with bleaching herbicide influenced the lipid toprotein ratio, membrane fluidity and structural organization ofthe chloroplasts [41] and affect the stability of the complexes[42]. In addition the present data emphasize that the degree ofcarotenoid depletion influences the fluorescence characteristicsand functions of both photosystems. The results are summarizedin Table 5. The changes in the low temperature fluorescence emis-sion spectra are registered even at a small decrease of the Car con-tent (25CarD) when the chlorophyll content is decreased only by12% (Tables 1 and 2). The fluridone induced changes in the mem-brane organization at 40CarD and 67CarD samples, influenced alsothe maximum quantum yield for primary photochemistry in DAS(UPo = Fv/Fm = kP/(kN + kP)) and in LAS ðU0Po ¼ F 0v=F 0m ¼ k0P=ðkNk0Nþk0PÞÞ (Tables 3 and 5), as a result of the influence on the photochem-ical and non-photochemical processes of PSII de-excitation.

The results from fluorescence quenching analyses show thatfluridone treatment causes a considerable decrease in q0P, which

Table 5Summary of the effect of different degree of carotenoid depletion (CarD) on thefunction of photosynthetic apparatus. Valuation Factor (VF) expressed as the ratio ofthe corresponding parameter value of treated to value of untreated plants: maximumquantum yield for primary photochemistry in DAS, UPo = kP/(kN + kP); maximumquantum yield for primary photochemistry, U0Po ¼ k0P=ðk

0N þ k0PÞ; photochemical

quenching coefficient in LAS, q0P; actual quantum yield for primary photochemistryin LAS, U0Ps; ratio of the rate constants for non-photochemical quenching events in LASversus DAS, k0N=kN; Amplitude of P700 oxidation, P700+; ratio of slow to totalamplitude of P700 re-reduction IS/I; amplitude of the initial oxygen burst undercontinuous irradiation of thylakoid membranes, A; oxygen flash yield after the thirdflash Y3. For details see Section 2 (– not measured).

Parameter Valuation Factor (VF)

25CarD 40CarD 65CarD

UPo = Fv/Fm 0.98 0.63 0.49U0Po ¼ F 0v=F 0m 0.94 0.65 0.19U0Ps ¼ ðF

0m � F 0sÞ=F 0m 0.70 0.38 0.10

q0P ¼ ðF0m � F 0sÞ=F0v 0.75 0.59 0.52

k0N=kN ¼ Fm=F 0m 0.97 0.78 0.77P700+ 0.99 0.76 0.69IS/I 0.84 0.45 0.25A 0.64 0.37 –Y3 0.29 0.15 –

indicates larger amount of closed PSII reaction centers or a de-crease in the fraction of the excitation energy used for photochem-istry. To estimate the relative fraction of reduced QA we calculatethe parameter V 0sðV

0s ¼ 1� q0P ¼ Q�A=QA totalÞ. The increase in V 0s

and therefore the decrease in the parameter q0P are registered evenat 25CarD, which indicates a rise of the fraction of closed PSII reac-tion centers (Table 3). Blocking of the electron transfer from QA toQB, i.e. an increase in the fraction of QB-non-reducing centers prob-ably leads to the observed accumulation of reduced QA. The datashow that even 25CarD influence on the amount of QB-non-reduc-ing centers, i.e. the number of these centers increases in compari-son to the untreated plants (Tables 3 and 5). The actual quantumyield for primary photochemistry in LAS, U0Ps, is also influencedeven at 25CarD. The changes in U0Ps at 25CarD are mainly from de-crease of the q0P, as a result of an increased number of the QB-non-reducing centers, while the changes at 65CarD are mainly fromstrong decrease in the maximum quantum yield for primary pho-tochemistry in LAS (Tables 3 and 5). Further depletion of the Car(40CarD and 65CarD) influences the non-photochemical de-excita-tion processes ðk0N=kNÞ. It was previously reported that alteration inphotosystem stoichiometry, antenna sizes and xanthophyll cycleactivity also influences these processes [43,44]. Havaux et al.[45], using mutants of Arabidopsis thaliana deficient in the xantho-phylls, have shown that the destabilization of LHCII and decreaseof its content leads to diminishing of the quantum efficiency of PSIIand non-photochemical de-excitation processes. Having in mindthese observations, it could be supposed that changes in k0N=kN

and chlorophyll fluorescence emission of PSII at 77 K (Table 2and Fig. 1) of thylakoid membranes from fluridone treated plantsare due to alterations in PSII antenna complexes organization.

The assumption for increasing of the fraction of reduced QA

could explain the loss of the functionally active PSII centers (corre-lated with parameter A, Table 4) after Car depletion. On the otherhand, the oxygen-evolving rate, corresponding to an electron flowfrom oxygen evolving complex to the plastoquinone pool andkinetics of P680+ reduction, depends on Si state transitions[46,47]. Our results indicate that small changes in the Car contentaffect the S0–S1 state distribution in darkness and lead to rise in themisses and double hits (Table 4). The oxidation state of Mn clustersin S0 (Mn2+, Mn3+, Mn4+, Mn4+) is lower by one oxidizing equivalentthan in S1 (Mn3+, Mn3+, Mn4+, Mn4+) [48]. The enhanced populationof centers in the S0 state (in darkness) was observed after fluridonetreatment. Thus, higher amount of Mn ions in Mn(II) oxidationstate (increased of the centers in S0) was observed in thylakoidmembranes with decreased Car content. On the other hand, Cardepletion leads to decrease in the ratio of the PSIIa to PSIIb centers(ratio A1/A2, Table 4), pointing to decrease of the centers evolvingoxygen through non-cooperative Kok’s mechanism (PSIIa), whichis characterized by the amplitude A1. Moreover, the decrease ofthe rate constants k1 and k2 of the two components of decay ofthe initial oxygen burst (Table 4) could be a consequence of thechanges on the acceptor side of PSII, which modify the interactionbetween QB and plastoquinone, and/or influence on the function ofOEC. Our previous investigation showed that structural organiza-tion of PSII supercomplex (degree of LHCII oligomerization)strongly influences the oxygen evolution [34]. Having in mind thisresult, it could be supposed that changes in the oxygen evolution(Tables 4 and 5) might also be a result from alterations in the LHCIIorganization as well as the changes in the lipid composition [12,49]of the membranes as a result of decreased Car content.

While changes in the function of OEC are registered in theplants with 25CarD there are no apparent alterations in the far-red induced P700 oxidation (Fig. 4). The decrease of the P700 oxi-dation is observed after 40CarD (Figs. 4 and 5), which could be aresult from changes in the organization of the PSI complex. Insupport of this statement is the 3 nm shift in the position of the

K. Dankov et al. / Journal of Photochemistry and Photobiology B: Biology 96 (2009) 49–56 55

fluorescence emitted from PSI in 77 K chlorophyll fluorescenceemission spectra (Fig. 2). The re-reduction kinetics of P700+ is de-scribed by two phases, which reveal either two different electrondonor systems or reduction of two different PSI types [50]. The firstphase of reduction is fast and the second is slow. The donor systemcapable of fast electron donation may be a reduced electron carrierin the cyclic electron transport, perhaps ferredoxin, whereas theslow donor could be a reduced pyridine nucleotide in chloroplaststroma [51,52]. Alternatively, the heterogeneity of P700+ reductioncan be explained with the suggestion of Albertsson [50] for theexistence of two different pools of PSI located in different domainsof thylakoid membranes. It is known that PSI from grana marginsand stroma lamellae are different in their ability to reduce ferre-doxin [53]. According to the model of Albertsson [50] the linearelectron transport occurs in the grana while cyclic electron trans-port is restricted to the stroma lamellae. Bukhov et al. [54] dis-cussed the heterogeneity of the kinetic of P700+ reduction afterfar-red oxidation in terms of the PSI-dependent electron transportin stroma lamellae and grana margins. The rapidly operating path-way could be driven by enzymes located in the stroma lamellae,whereas enzymes mediating the slow pathway are in grana thylak-oids [55]. Our data show that with decreasing of the Car content(more than 40CarD), diminishing of the slow component is ob-served (Fig. 5). This is an argument for a predominant role of theferredoxin-dependent pathway rather than for direct electrondonation from pyridine nucleotides or for a stronger influence onthe PSI in the grana margins than in the stroma lamellae.

In summary, the experimental results show that the various de-gree of Car depletion (25CarD, 40CarD and 65CarD) has differentinfluence on the yield of chlorophyll fluorescence, primary photo-chemistry, photosynthetic oxygen evolution and P700 photooxida-tion (Table 5). In plants with 25CarD the primary photochemistryin DAS and LAS (UPo and U0Po, respectively) and P700 oxidationare not influenced, while the actual quantum yield for primaryphotochemistry ðU0PsÞ and photosynthetic oxygen evolution are de-creased. The higher levels of Car depletion (40CarD and 65CarD)with concomitant structural changes in the photosynthetic appara-tus lead to: (i) decrease in both primary photochemistry and pho-tosynthetic oxygen evolution; (ii) inhibition of the photosyntheticoxygen evolution, which is stronger than the effect on the primaryphotochemistry in LAS and DAS and (iii) effects on the PSI photo-chemistry (P700 oxidation), which are smaller than the effect onthe PSII (UPo and U0Po). Especially important observation is thatthe inhibition of the functionally active PSII centers and modifica-tion of the still active centers, as a result from Car depletion in-duced structural changes in PSII supercomplex, which are morepronounced in PSIIa than in PSIIb centers.

5. Abbreviations

A amplitude of the oxygen burst under continuous irradia-tion

Car carotenoidsCarD carotenoid depletionChl chlorophyllDAS dark-adapted stateLAS light-adapted stateLHCII light-harvesting chlorophyll a/b protein complex of PSIIOEC oxygen evolving complexPSI photosystem IPSII photosystem IISi redox state i of the OECF 0s steady state chlorophyll fluorescence in LASFm maximum chlorophyll fluorescence in DASF 0m maximum chlorophyll fluorescence in LASF0 minimum chlorophyll fluorescence in DAS

F 00 minimum chlorophyll fluorescence in LASFv variable fluorescence in DASF 0v variable fluorescence in LASUPo maximum quantum yield for primary photochemistry in

DASU0Po maximum quantum yield for primary photochemistry in

LASU0Ps actual quantum yield for primary photochemistry in LASq0P photochemical quenching coefficient in LASkP rate constant of photochemical de-excitation processeskN rate constant of non-photochemical de-excitation pro-

cessesV 0s relative variable fluorescence in LAS

Acknowledgements

The authors thank to Prof. Y. Zeinalov from the Institute of Bio-physics, Bulgarian Academy of Sciences, for the critical reading ofthe manuscript and insightful comments. The work was supportedby contract B-1512/05 with National Science Fund. The authors aregrateful to Prof. N.P.A. Huner and Dr. A.G. Ivanov from the Univer-sity of Western Ontario, Canada for the possibility to use PAM fluo-rometer (H. Walz, Effeltrich, Germany, model PAM 101–103).Thanks are also to the colleagues from Department of Regulationof Plant Growth and Development, Institute of Plant Physiology,Bulgarian Academy of Sciences, for cultivate of the fluridone trea-ted plants.

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