Altered Turnover of b-Carotene and Chl a in ArabidopsisLeaves Treated with Lincomycin or NorflurazonKim Gabriele Beisel, Ulrich Schurr and Shizue Matsubara*IBG-2: Pflanzenwissenschaften, Forschungszentrum Julich, D-52425 Julich, Germany*Corresponding author: E-mail, s.matsubara@fz-juelich.de; Fax, +49-2461-61-2492(Received March 2, 2011; Accepted May 25, 2011)

Interactions between b-carotene (b-C) and Chl a turnoverwere investigated in relation to photoinhibition and D1 pro-tein turnover in mature leaves of Arabidopsis (Arabidopsisthaliana) by 14CO2 pulse–chase labeling. Following a 2 htreatment of leaves with water, lincomycin (Linco; an inhibi-tor of chloroplast protein synthesis) or norflurazon (NF; aninhibitor of carotenoid biosynthesis at phytoene desatur-ation) in the dark, 14CO2 was applied to the leaves for30 min under control light (CL; 130 mmol photons m–2 s–1)conditions, followed by exposure to either CL or high light(HL; 1,100 mmol photons m–2 s–1) in ambient CO2 for up to6 h. Under both light conditions, 14C incorporation wasstrongly decreased for Chl a and moderately suppressedfor b-C in Linco-treated leaves, showing a marked declineof PSII efficiency (Fv/Fm) and b-C content compared withwater-treated leaves. Partial inhibition of carotenoid biosyn-thesis by NF caused no or only a minor decrease in Fv/Fm andChl a turnover under both conditions, while the b-C contentsignificantly declined and high 14C labeling was found forphytoene, the substrate of phytoene desaturase. Together,the results suggest coordinated turnover of Chl a and D1,but somewhat different regulation for b-C turnover, inArabidopsis leaves. Inhibition of carotenoid biosynthesis byNF may initially enhance metabolic flux in the pathway up-stream of phytoene, presumably compensating for shortsupply of b-C. Our observations are also in line with thenotion that HL-induced accumulation of xanthophylls mayinvolve a precursor pool which is distinct from that for b-Cturnover.

Keywords: Arabidopsis thaliana � b-Carotene � Chl �

Lincomycin � Norflurazon � Photoinhibition.

Abbreviations: A, antheraxanthin; b-C, b-carotene; CL, con-trol light (130mmol photons m–2 s–1); Fv/Fm, maximal PSIIefficiency in dark-adapted leaves; HL, high light (1,100 mmolphotons m–2 s–1); Linco, lincomycin; Lut, lutein; Neo, neox-anthin; NF, norflurazon; Phy, phytoene; V, violaxanthin; Z,zeaxanthin.

Introduction

Light is essential for photosynthesis but it can also cause oxi-dative damage to the photosynthetic apparatus when the lightenergy absorbed by Chl a molecules cannot be utilized forphotochemical reactions. A range of photoprotective mechan-isms exist in and around pigment–protein complexes of photo-synthetic membranes (thylakoids) to minimize photooxidativedamage resulting from formation of triplet-state Chl and singletO2 (Demmig-Adams 1990, Noctor and Foyer 1998, Muller et al.2001, Maeda and DellaPenna 2007). Nevertheless, PSII, the siteof water oxidation and plastoquinone reduction, undergoeslight-induced inactivation even under low irradiance, whichmanifests itself as irreversible damage to the reaction centerpolypeptide D1 (Tyystjarvi and Aro 1996, Keren et al. 1997).Damaged D1 protein of photoinactivated PSII is replaced by anew copy through an elaborate repair cycle operating betweenstacked grana and non-stacked stroma regions of thylakoids(Melis 1999, Baena-Gonzalez and Aro 2002). The D1 and D2polypeptides of the PSII reaction center bind six Chl a, twopheophytin, two plastoquinone and two b-carotene (b-C) mol-ecules (Kobayashi et al. 1990). Whereas Chl a, pheophytin andplastoquinone are engaged in the PSII electron transfer asredox-active cofactors, b-C is thought to play a photoprotectiverole (Tracewell et al. 2001, Telfer 2005). It has been suggestedthat translation elongation and membrane insertion of a newD1 protein during the D1 repair cycle requires assembly part-ners (Zhang et al. 1999) as well as ligation of Chl a (Kim et al.1994, He and Vermaas 1998) and b-C (Trebst and Depka 1997).Whenever the repair and turnover of D1 cannot keep pace withthe rate of photodamage, e.g. upon an increase in irradiance orunder stress conditions, the overall efficiency of PSII declines(‘photoinhibition’).

In parallel with the continuous D1 turnover in the PSII re-action center in the light, interconversions between epoxidizedand de-epoxidized xanthophylls (xanthophyll cycle) take placein thylakoids, catalyzed by two enzymes, violaxanthin (V)de-epoxidase and zeaxanthin (Z) epoxidase (Jahns et al.2009). Light-induced acidification of thylakoid lumen activates

Plant Cell Physiol. 52(7): 1193–1203 (2011) doi:10.1093/pcp/pcr069, available online at www.pcp.oxfordjournals.org! The Author 2011. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.All rights reserved. For permissions, please email: journals.permissions@oup.com

1193Plant Cell Physiol. 52(7): 1193–1203 (2011) doi:10.1093/pcp/pcr069 ! The Author 2011.

Regu

larP

aper

at Forschungszentrum

Juelich Gm

bH, Z

entralbibliothek on July 13, 2011pcp.oxfordjournals.org

Dow

nloaded from

V de-epoxidase which converts V molecules released fromlight-harvesting antenna complexes into antheraxanthin (A)and Z. The reactions are reversed by the activity of Z epoxidase,which becomes evident when V de-epoxidase is inactive in thedark or under non-stressful light intensities. Accumulation ofZ via operation of the xanthophyll cycle enhances non-photochemical quenching of singlet-excited Chl (Demmig-Adams 1990, Muller et al. 2001) and provides antioxidativeprotection (Havaux and Niyogi 1999) under strong illu-mination. Consistent with these photoprotective functions,sunlit leaves typically contain larger amounts of the xantho-phyll cycle pigments (V + A + Z) than leaves in shaded envir-onments (e.g. Demmig-Adams and Adams 1992, Matsubaraet al. 2009).

De novo synthesis of V + A + Z (especially Z) as well as b-Cand/or lutein (Lut) has been observed in leaves in response tohigh light exposure and associated with up-regulation of carot-enoid biosynthesis to enhance photoprotection (e.g. Garcıa-Plazaola et al. 2002, Forster et al. 2009). Recently, we havedemonstrated continuous turnover of b-C and Chl a inArabidopsis (Arabidopsis thaliana) leaves by 14CO2 pulse–chase labeling (Beisel et al. 2010). Based on rapid 14C incorpor-ation into these PSII core complex pigments, but not inlight-harvesting antenna pigments (xanthophylls and Chl b),turnover of b-C and Chl a observed under both stressful andnon-stressful light conditions was regarded as part of the D1repair cycle. Since Z is synthesized by b-ring hydroxylation ofb-C, a direct link between b-C turnover during the D1 repaircycle and stress-induced Z accumulation has been proposed forChlamydomonas reinhardtii (Depka et al. 1998). An importantimplication of these previous observations is that carotenoidmetabolism in chloroplasts can sense and respond to environ-mental changes to meet photoprotective demand for differentcarotenoids. In support of this notion, increased transcriptabundance of b-ring hydroxylase genes, the products ofwhich predominantly catalyze hydroxylation of b-C to Z (Kimet al. 2009), has been reported for Arabidopsis leaves shortly(�1 h) after high light exposure (Rossel et al. 2002, Cuttrisset al. 2007).

If degradation and synthesis of b-C and Chl a play a part inthe D1 repair cycle in leaves, turnover of these pigments maybe regulated in conjunction with concurrent turnover of D1protein. In order to gain insights into interactions betweenturnover of b-C, Chl a and D1, we have conducted 14CO2

pulse–chase labeling experiments in Arabidopsis leaves fol-lowing treatments with inhibitors of D1 or b-C synthesis.Lincomycin (Linco), an inhibitor of chloroplast protein syn-thesis, arrests translation of the plastid-encoded psbA geneproduct (D1), and thus repair of photoinactivated PSII, resultingin reduced PSII efficiency in leaves (Tyystjarvi and Aro 1996, Leeet al. 2001). Synthesis of b-C can be inhibited by norflurazon(NF) which impairs the catalytic activity of phytoene (Phy)desaturase upstream of b-C. Carotenoid (b-C) depletion byincubation with NF is known to diminish D1 as well as photo-synthetic activity in green algae (Sandmann et al. 1993,

Trebst and Depka 1997). Also in higher plants, NF treatmentleads to impaired PSII efficiency, decreased contents of D1 andother Chl-binding protein complexes of PSII, and, ultimately,bleaching (Markgraf and Oelmuller 1991, Corona et al. 1996,Xu et al. 2000, Welsch et al. 2003). After pre-treatment withthese inhibitors and a chase period of up to 6 h under differentlight conditions, pigments were extracted from leaves and theradioactivity of each 14C-labeled pigment was measured. Theresults are discussed in terms of (i) whether the turnover pro-cesses of b-C, Chl a and D1 are coupled with each other inmature Arabidopsis leaves; and (ii) how carotenoid synthesisresponds to NF-induced restriction of metabolic flux in thepathway.

Results

Maximal PSII efficiency

After detaching leaves from Arabidopsis plants grown under130 mmol photons m–2 s–1 (control light, CL), petioles wereimmediately put in water (Control), 3 mM Linco or 70mM NFsolution and the leaves were kept for 2 h in the dark with air-flow forcing transpiration. The leaves were then floated onwater or diluted inhibitor solutions (1 mM Linco or 10 mMNF) with the adaxial surface facing the air, and placed under130 mmol photons m–2 s–1 (CL!CL treatment) or 1,100 mmolphotons m–2 s–1 (CL!HL treatment) starting at 0 h (Fig. 1).Measurements of Chl a fluorescence were performed at differ-ent time points during the light treatments. The maximal PSIIefficiency (Fv/Fm) remained high in water-treated leaves andNF-treated leaves under CL!CL, whereas the correspondingleaves in CL!HL showed a significant decrease (Fig. 1A, C).While the initial (up to 0.5 h) rapid decrease under CL!HL wassimilar in water-treated leaves and NF-treated leaves, the sub-sequent slow decrease in Fv/Fm was more pronounced inNF-treated leaves. The leaves treated with Linco exhibited asignificant and almost linear reduction of Fv/Fm from 0.84 to0.62 over 6 h under CL!CL (Fig. 1B), whereas Fv/Fm declinedrapidly to <0.6 in the first 30 min and to almost zero after 6 hunder CL!HL conditions.

Chl and carotenoid composition

Chl and carotenoid composition was analyzed in leaves dur-ing the Fv/Fm measurements under CL!CL (Fig. 2) andCL!HL conditions (Fig. 3). In both conditions, neither Chl anor Chl b content changed significantly in leaves of thethree treatments throughout the 6 h experiment (Figs. 2A,D, G, 3A, D, G).

Under CL!CL, V was generally the major xanthophyll cyclepigment in leaves (Fig. 2B, E, H); only traces of A and Z could bedetected. Except for a small and transient decrease in V inwater-treated leaves shortly after the dark to CL transfer at0 h, the levels of the xanthophyll cycle pigments remained prac-tically unchanged during the experiment and the values werecomparable between the leaves treated with water, Linco and

1194 Plant Cell Physiol. 52(7): 1193–1203 (2011) doi:10.1093/pcp/pcr069 ! The Author 2011.

K. G. Beisel et al.

at Forschungszentrum

Juelich Gm

bH, Z

entralbibliothek on July 13, 2011pcp.oxfordjournals.org

Dow

nloaded from

Control Lincomycin Norflurazon

100

80

60

40

20

80

60

40

20

0

180

150

120

90

60

30

A

B

C

D

E

F

G

H

I

2 4 6 2 4 6 2000 4 6

Time [h]

Chl a

Ch

l a

, C

hl

b (

mmol

m-2

)V

, A

, Z

(m

mol

mol

-1 C

hl a

)L

, b-

C,

N (

mm

ol m

ol-1

Chl

a)

Chl b

V+A+ZVAZ

N

Lb-C

Fig. 2 Changes in Chl and carotenoid contents of Arabidopsis leaves under CL!CL conditions. Leaves were pre-treated with (A–C) water(Control), (D–F) 3 mM lincomycin or (G–I) 70 mM norflurazon for 2 h in the dark prior to 0 h. Chl contents are expressed per unit of leaf area(mmol m–2). Carotenoid contents are given relative to the Chl a content (mmol mol–1 Chl a). Asterisks below the symbols indicate significantdifferences (P< 0.05) compared with 0 h of the same pigment for each treatment. For the xanthophyll cycle pigments, only the data of the totalV + A + Z were statistically tested. All data are means ± SE (n = 3). Error bars are shown when they are larger than the symbols.

Control Lincomycin NorflurazonBA C

0.8 0.8

0.6 0.6

0.4

Fv/

Fm

Fv/

Fm

0.2

0.4

0.2CL HL

CL CL

0.0 0.0

0 2 4 6 0 2 4 6 0 2 4 6

Time [h]

Fig. 1 The maximal PSII efficiency (Fv/Fm) measured in leaves of Arabidopsis under different light conditions. Leaves were detached from plantsgrown under 130 mmol photons m–2 s–1 (CL) and treated with (A) water (Control), (B) 3 mM lincomycin or (C) 70 mM norflurazon for 2 h in thedark. Then, they were floated on water or diluted inhibitor solutions (1 mM lincomycin or 10 mM norflurazon), with the adaxial surface facing theair, and placed under 130 mmol photons m–2 s–1 (CL!CL, filled circles) or 1,100 mmol photons m–2 s–1 (CL!HL, open circles) at 0 h. All data aremeans ± SE (n = 5). Error bars are shown when they are larger than the symbols.

1195Plant Cell Physiol. 52(7): 1193–1203 (2011) doi:10.1093/pcp/pcr069 ! The Author 2011.

Turnover of b-carotene and Chl a in leaves

at Forschungszentrum

Juelich Gm

bH, Z

entralbibliothek on July 13, 2011pcp.oxfordjournals.org

Dow

nloaded from

NF. The initial levels of Lut, neoxanthin (Neo) and b-C were alsosimilar in the three treatments (Fig. 2C, F, I). However, whileLut and Neo remained nearly constant, the b-C contents sig-nificantly decreased by 6 h in Linco-treated leaves and by 3 h inNF-treated leaves.

The carotenoid composition changed substantially in theleaves of all three treatments under CL!HL conditions(Fig. 3). In the first 30 min, water-treated leaves quickly accu-mulated Z and some A at the expense of V via operation ofthe xanthophyll cycle (Fig. 3B); the extent of de-epoxidationwas 70% (i.e. 70% of V + A + Z in the form of Z or A) at 0.5 h.The highest level of de-epoxidation (90%) was reached at 3 h,concomitant with a significant increase (+50% compared with0 h) in the total amount of V + A + Z. Thereafter, no further de-epoxidation or increase in V + A + Z was observed. Linco-treated leaves showed similar but slower changes comparedwith water-treated leaves (Fig. 3E); de-epoxidation progressedmore gradually after the first 30 min and V + A + Z initiallydecreased and then increased by 30% between 3 and 6 h. Theoperation of the xanthophyll cycle was also evident in

NF-treated leaves under CL!HL (Fig. 3H). The time courseof de-epoxidation in NF-treated leaves was very similar to thepattern in water-treated leaves. The increase in V + A + Z wasalso found in these leaves between 0.5 and 3 h, but it was muchsmaller than in water-treated leaves.

Regardless of the inhibitor treatments, Neo remained un-changed under CL!HL conditions (Fig. 3C, F, I). The vari-ations in Lut were mostly not significant, with the exceptionof the somewhat higher values (+15% compared with 0 h) inwater-treated leaves at 3 and 6 h (Fig. 3C). This increase in Lutcoincided with the increase in V + A + Z in these leaves(Fig. 3B). As seen under CL!CL, the b-C contents werestable in water-treated leaves under CL!HL (Fig. 3C) whereasboth Linco-treated leaves and NF-treated leaves showed a sig-nificant decrease in b-C (Fig. 3F, I). The levels of b-C decreasedmore rapidly and/or strongly in CL!HL than in CL!CL,with the total reduction under CL!HL after 6 h being –32and –27% in Linco-treated leaves and NF-treated leaves, re-spectively, compared with –6 and –12% in the correspondingleaves under CL!CL.

Control Lincomycin Norflurazon

100

80

60

40

20

80

60

40

20

0

180

150

120

90

60

30

A

B

C

D

E

F

G

H

I

2 4 6 2 4 6 2000 4 6

Time [h]

Chl a

Ch

l a

, C

hl

b (

mmol

m-2

)V

, A

, Z

(m

mol

mol

-1 C

hl a

)L

, b-

C,

N (

mm

ol m

ol-1

Chl

a)

Chl b

V+A+ZVAZ

N

Lb-C

Fig. 3 Changes in Chl and carotenoid contents of Arabidopsis leaves under CL!HL conditions. For a description of treatments and dataanalysis, see the legend to Fig. 2.

1196 Plant Cell Physiol. 52(7): 1193–1203 (2011) doi:10.1093/pcp/pcr069 ! The Author 2011.

K. G. Beisel et al.

at Forschungszentrum

Juelich Gm

bH, Z

entralbibliothek on July 13, 2011pcp.oxfordjournals.org

Dow

nloaded from

14C-Labeled pigmentsPulse–chase labeling experiments were conducted with 14CO2

in order to infer synthesis and degradation of photosyntheticpigments in Arabidopsis leaves in the presence of Linco or NF.Following the treatment with either water or the inhibitors(3 mM Linco or 70mM NF) for 2 h in the dark, leaves weresubjected to a 30 min pulse application of 14CO2 under CL,with their petioles incubated in the corresponding solutions(water, 1 mM Linco or 10 mM NF). Then, leaves were placedunder CL!CL or CL!HL conditions in ambient air for differ-ent durations (chase) while floating on water, Linco (1 mM) orNF (10 mM) solution. Incorporation of 14C into photosynthetic

pigments was examined by radio-HPLC analysis (Beisel et al.2010), and the radioactivity of each pigment was expressedrelative to the Chl a content.

For all treatments, rapid and strong incorporation of 14C wasdetected in Chl a (Fig. 4A, B) but not in Chl b (data not shown).The radiosignal of Chl a did not differ significantly betweenCL!CL and CL!HL conditions, even when the leaves weretreated with Linco or NF. The 14C levels of Chl a were compar-ably high in water-treated leaves and NF-treated leaves through-out the chase; only the water-treated leaves in CL!CL showeda tendency towards increasing radioactivity for Chl a at the endof the experiment. In contrast, much less radioactivity (�40

400 H2O

LinconmycinNorflurazon

14C

in C

hl a

[Net

Bq

mg C

hl a

-1]

14C

in b

-C[N

et B

q mg

Chl

a-1

]

14C

in P

hy[N

et B

q mg

Chl

a-1

]

A

CL CL CL HL

300

200

100

100 C

80

60

40

20

E

B

D

F200

150

100

50

0

0 2 4 6 0 2 4 6

Time [h]

Fig. 4 Changes in 14C radioactivity incorporated in (A and B) Chl a, (C and D) b-carotene and (E and F) phytoene under CL!CL or CL!HLconditions. Detached leaves were pre-treated with water (Control, filled circles), 3 mM lincomycin (open triangles) or 70 mM norflurazon (opensquares) for 2 h in the dark. Subsequently, 14CO2 was applied to the leaves under the CL condition for 30 min (pulse period). At the end of thepulse period (at 0 h), the leaves were transferred to ambient CO2 and chase was performed under CL (A, C and E) or HL (B, D and F) for up to 6 h.The 14C radioactivity was normalized to the Chl a content measured in the same samples (Bq mg–1 Chl a). For each time point, asterisks and plussigns below the symbols indicate significant differences (P< 0.05) between control and lincomycin-treated leaves, or between control andnorflurazon-treated leaves, respectively. All data are means ± SE (n = 3).

1197Plant Cell Physiol. 52(7): 1193–1203 (2011) doi:10.1093/pcp/pcr069 ! The Author 2011.

Turnover of b-carotene and Chl a in leaves

at Forschungszentrum

Juelich Gm

bH, Z

entralbibliothek on July 13, 2011pcp.oxfordjournals.org

Dow

nloaded from

and 50% in CL!CL and CL!HL, respectively) was measured inthe Linco-treated leaves.

Also b-C showed similarly high 14C signals under both lightconditions (Fig. 4C, D), with no or little labeling detected forxanthophylls (data not shown). The 14C signal of b-C reachedmaximal levels after a 30 min chase, followed by a decrease orno substantial change until 6 h. In general, less 14C was incor-porated in b-C in the inhibitor-treated leaves compared withwater-treated leaves. Despite the treatment with NF, b-C wasclearly labeled with 14C in the Arabidopsis leaves; underCL!HL a significant reduction in the 14C-labeled b-C wasobserved with Linco but not with NF (Fig. 4D).

However, a prominent peak emerged in the radiograms ofNF-treated leaves at the position of Phy (Fig. 5), the substrate ofPhy desaturase, confirming the effect of our NF treatment.While only marginal levels of 14C radioactivity were measuredfor Phy in water-treated leaves and Linco-treated leaves, strik-ingly high 14C labeling of Phy was found in NF-treated leavesunder CL!CL as well as CL!HL conditions (Fig. 4E, F). Theradiosignal of Phy in NF-treated leaves (>100 Net Bq mg–1 Chla) was higher than the highest radiosignal of b-C measured inthese experiments (�80 Net Bq mg–1 Chl a). The 14C ratio ofPhy : Chl a was about 1 : 2 in NF-treated leaves, with the valuesapproaching 1 : 1 in CL!HL after 6 h. For comparison, the 14Cratio of b-C : Chl a was between 1 : 3 and 1 : 5 in water-treatedleaves, between 1 : 3 and 2 : 3 in Linco-treated leaves and be-tween 1 : 4 and 1 : 5 in NF-treated leaves. When the 14C signalsof b-C and Phy were added together, NF-treated leaves had a47% higher signal, and Linco-treated leaves had a 49% lowersignal, compared with water-treated leaves already at 0 h. Ofthe three treatments, Linco-treated leaves had the lowest total14C radioactivity in the sum of Chl a, b-C and Phy at anytime point, which was about 60% lower than in water-treatedleaves.

Discussion

Effects of lincomycin on pigment turnover

The D1 protein of the PSII reaction center undergoes a con-tinuous repair cycle at all light intensities (Tyystjarvi and Aro1996), going through photoinactivation, PSII disassembly, D1degradation, insertion of a newly synthesized D1 and PSII re-assembly (Baena-Gonzalez and Aro 2002). The antibiotic Lincoinhibits protein synthesis in chloroplasts, thereby disrupting,amongst others, the D1 repair cycle and leading to accumula-tion of photoinactivated PSII (Tyystjarvi et al. 1992, Lee et al.2001). This explains the reduced maximal PSII efficiency inLinco-treated leaves even under CL!CL (–25% after 6 h;Fig. 1B), revealing the extent of PSII photoinactivation inleaves of CL-grown Arabidopsis plants under non-stressful CLconditions. Obviously, the repair cycle could compensate forthis level of D1 damage in water-treated leaves under CL!CL(Fig. 1A). However, transfer to HL dramatically accelerated therate of PSII photoinactivation in all three treatments (Fig. 1A),

concomitant with the operation of the xanthophyll cycle andassociated photoprotective mechanisms (Fig. 3B, E, H). UnderCL!HL conditions, the rate of photoinactivation exceeded therepair capacity of the CL-acclimated plants, resulting in a

Fig. 5 Radio-HPLC analysis for identification of [14C]phytoene.Chromatograms recorded at (A) 440 nm and (B) 286 nm. Shown arethe pigment sample extracted from a norflurazon-treated leaf labeledwith 14CO2 and then exposed to HL for 6 h (leaf), the same sampleafter addition of 1mg of Z- and E-phytoene standard (leaf + standard)and 1 mg Z- and E-phytoene standard (standard). Peak 1, Chl a; peak 2,Z-phytoene; peak 3, E-phytoene; peak 4, b-carotene. AU, arbitraryunits. (C) Simultaneous radiogram of 14C-labeled compounds in theleaf sample. Dotted lines indicate the expected positions of pigmentpeaks in the radiogram, with a 20 s offset compared with the corres-ponding peaks in the chromatograms due to the sequential detectionby the UV-visible detector and the radiodetector.

1198 Plant Cell Physiol. 52(7): 1193–1203 (2011) doi:10.1093/pcp/pcr069 ! The Author 2011.

K. G. Beisel et al.

at Forschungszentrum

Juelich Gm

bH, Z

entralbibliothek on July 13, 2011pcp.oxfordjournals.org

Dow

nloaded from

decline in Fv/Fm, which manifested itself most strikingly inLinco-treated leaves (Fig. 1B).

Much less 14C was incorporated in both Chl a and b-C inLinco-treated leaves compared with water-treated leaves(Fig. 4), indicating reduced de novo synthesis of these pigmentsafter the Linco treatment. The reduction of 14C incorporation inLinco-treated leaves was more pronounced for Chl a than forb-C, suggesting closer interactions between D1 protein and Chla turnover. Since Chl a molecules and some of the Chl precur-sors can become harmful photosensitizers unless they arebound to proteins (Meskauskiene et al. 2001), continuous syn-thesis and degradation of Chl a may need to be coordinatedwith the D1 repair cycle. While some secondary effects of Lincocannot be ruled out (Fiekers et al. 1979), the low 14C signal ofChl a measured already at 0 h (Fig. 4A, B) suggests quickdown-regulation of Chl a synthesis upon inhibition of D1 pro-tein synthesis. Whether this Linco-induced down-regulation ofChl a turnover is mediated by retrograde signaling is notknown. Considering the immediacy of down-regulation, how-ever, the earliest events do not seem to depend on reactionsstarting from gene transcription.

The total Chl a content did not change in Linco-treatedleaves under CL!CL conditions (Fig. 2D), and there wasonly a small, statistically non-significant decrease in Chl aunder CL!HL at 3 h (Fig. 3D) despite severe PSII photoinhibi-tion (Fig. 1B). Given that no more than a few percent of Chl amolecules are bound in PSII reaction centers in leaves, changesin Chl a content during photoinactivation and repair are prob-ably difficult to detect by measuring the total Chl a amount.Judging from the low but steady levels of Chl a radiosignal inspite of the acute PSII photoinhibition in Linco-treated leavesafter 6 h in CL!HL (Figs. 4B, 1B), both synthesis and degrad-ation of Chl a seem to slow down when the D1 protein turnoveris inhibited. While Chl recycling (Vavilin and Vermaas 2007)may maintain the steady-state 14C level of Chl a, operation ofChl a recycling in photoinactivated PSII is unlikely unless thereis concurrent operation of D1 repair. Parallel down-regulationof Chl a and D1 turnover suggests possible enzymatic controlof Chl a degradation during the PSII repair cycle, although thisdoes not preclude photooxidation of Chl a under excess light.In addition to thermal dissipation in antenna complexes, strongquenching in photoinactivated PSII (Matsubara and Chow2004) could reduce formation of Chl a triplet states and theresulting production of singlet O2 to restrict oxidative degrad-ation of Chl a.

The effects of Linco on b-C turnover were similar to thoseobserved for Chl a, albeit not as large (Fig. 4C, D). Thus, cou-pling between b-C and D1 protein turnover may not be as tightas between Chl a and D1. This is in line with our previousfinding of contrasting HL-acclimatory responses of Chl a andb-C turnover in Arabidopsis leaves; acclimation to HL signifi-cantly enhanced Chl a turnover, as is known for D1 turnover(Tyystjarvi et al. 1992), while reducing that of b-C (Beisel et al.2010). Unlike Chl a, free b-C molecules can accumulate in mem-branes to confer photoprotection, as can be seen in leaves of

field-grown plants (Verhoeven et al. 1999, Matsubara et al.2003). Thus, synthesis and degradation of b-C may be subjectto regulatory mechanisms different from those of the D1 repaircycle.

In contrast to the small change in Chl a, the marked de-crease of the b-C content in Linco-treated leaves in CL!HL at3 h (–15%) and 6 h (–30%) (Fig. 3F) suggests enhanced b-Cdegradation, probably through photooxidation (Tracewellet al. 2001) after pronounced photoinhibition (Fig. 1B). Onemay ask whether b-C turnover in non-stressful conditions, likein water-treated leaves under CL!CL (Fig. 1A), also reflectsbleaching of this pigment in PSII or is controlled by carotenoidcleavage enzymes (Auldridge et al. 2006). Hydroxylation of b-Creleased from photoinactivated PSII during the repair cycle,giving rise to an increase in Z under HL stress as proposed forChlamydomonas (Depka et al. 1998), is an alternative scenarioto explain the observed b-C decrease concomitant with an in-crease in Z and V + A + Z (Fig. 3E, F) in Linco-treated leaves.However, the decreasing radiosignal of b-C in Linco-treatedleaves and water-treated leaves after 6 h under CL!HL (Fig.4D) did not result in 14C labeling of Z (data not shown), sug-gesting that hydroxylation of b-C to Z during b-C turnover maynot play a predominant role in Arabidopsis (Beisel et al. 2010).

Effects of norflurazon on pigment turnover

The herbicide NF inhibits carotenoid biosynthesis at the step ofPhy desaturation upstream of b-C. In contrast to obviousbleaching of leaves after long-term application of NF duringplant cultivation (e.g. Dalla Vecchia et al. 2001), our treatmentby feeding detached leaves with a 70 mM NF solution for 2 hdid not entirely stop 14C incorporation into b-C (Fig. 4C, D);longer (overnight) treatment with NF did not improve the in-hibitory effect (data not shown). The amount of NF taken up byleaves during these treatments was apparently not enough tosaturate the cofactor- (plastoquinone; Norris et al. 1995) bind-ing site of Phy desaturase to bring about full inhibition of theenzyme (Breitenbach et al. 2001). Nevertheless, the high 14Clabeling of Phy in NF-treated leaves (Fig. 4E, F), together witha decrease in b-C content under both CL!CL and CL!HLconditions (Figs. 2I, 3I), demonstrates some effects of theinhibitor.

Inhibition of b-C synthesis by NF resulted in loss of PSIIactivity and D1 protein in Chlamydomonas under high light,which has been interpreted as evidence for interrelationshipsbetween b-C and D1 protein turnover: photobleaching of b-C isthought to trigger D1 degradation, while the subsequent D1protein assembly in functional PSII seems to require b-C syn-thesis (Trebst and Depka 1997). In our experiments withNF-treated Arabidopsis leaves under CL!CL, partial inhibitionof b-C synthesis, causing approximately 15% reduction in b-Cafter 3 h (Fig. 2I), had no obvious impact on Chl a turnover(Fig. 4A) or photoinhibition (Fig. 1C), supporting loose cou-pling of b-C turnover with Chl a and D1 turnover, as was dis-cussed above for the Linco experiments. This is also in line with

1199Plant Cell Physiol. 52(7): 1193–1203 (2011) doi:10.1093/pcp/pcr069 ! The Author 2011.

Turnover of b-carotene and Chl a in leaves

at Forschungszentrum

Juelich Gm

bH, Z

entralbibliothek on July 13, 2011pcp.oxfordjournals.org

Dow

nloaded from

the recent finding in Synechocystis sp. PCC 6803 mutant cells,which are completely depleted of carotenoid pigments (Sozeret al. 2010); these mutant cells contain readily detectable levelsof D1 and PSII core assembly intermediates (lacking CP43 orboth CP43 and CP47) but no dimeric PSII and only a trace ofmonomeric PSII, indicating a role for b-C in the PSII core com-plex assembly.

On the other hand, it is difficult to reconcile the NF-inducedsignificant loss of b-C (Fig. 2I) with zero PSII photoinhibitionunder CL!CL (Fig. 1C) if b-C is needed for the D1 repair cycleeven if not directly at the step of D1 protein synthesis andinsertion. The following possibilities can be considered to ex-plain these observations: (i) Phy can replace b-C in PSII; (ii) PSIIreaction centers lacking b-C are rapidly degraded and do notaccumulate; or (iii) not all newly synthesized b-C molecules areneeded for D1 repair. Disappearance of PSII by deletion of thePhy desaturase gene in Synechocystis sp. PCC 6803 (Bautistaet al. 2005), accumulating Phy as the only carotenoid pigment,rejects (i). As NF-treated leaves showed photoinhibition(Fig. 1C) when the decrease in b-C was more pronouncedunder CL!HL (Fig. 3I), rapid degradation of photoinactivatedPSII reaction centers in the absence of b-C molecules, as statedin (ii), is rather unlikely. The presence of a b-C pool not requiredfor D1 repair and PSII activity, as assumed in (iii), could explainthe lack of a short-term effect of NF treatment on Chl a turn-over and Fv/Fm under CL!CL conditions, although 15% of theb-C pool in non-stressed leaves appears too large to be dispens-able for PSII activity.

Notably, we found about a 50% increase of 14C radioactivityin the sum of b-C and Phy (especially the latter) in NF-treatedleaves already at 0 h (Fig. 4C–F). These results are similar to theprevious report in leaves of Capsicum annuum showing markedaccumulation of Phy and increased total carotenoid levels(twice as high as the levels in control leaves when Phy isincluded) after 48 h of NF treatment (Simkin et al. 2003).Enhanced labeling of b-C + Phy found in Arabidopsis leavesshortly after NF treatment may suggest rapid up-regulation ofcarbon flux down the carotenoid biosynthetic pathway, per-haps via a metabolic feedback mechanism (Cazzonelli andPogson 2010). Activation of the Phy desaturase gene promoter,presumably by end-product regulation, has been documentedin tobacco seedlings grown on an NF-containing medium(Corona et al. 1996). Our data in Fig. 4 also point to compen-satory up-regulation, but upstream of Phy, which may precedethe transcriptional activation of the Phy desaturase gene. It hasbeen shown in Arabidopsis seedlings that gene transcription ofPhy synthase, the first committed and rate-limiting enzyme ofcarotenoid biosynthesis providing the substrate for Phy desa-turase, does not increase in response to NF (Welsch et al. 2003).If the situation is the same in mature leaves, the strong accu-mulation of Phy in NF-treated leaves may involve post-transcriptional enhancement of Phy synthase or up-regulationfurther upstream of the biosynthetic pathway.

Alternatively, a strong 14C signal of Phy in NF-treated leavesmay reflect high stability of Phy molecules (Simkin et al. 2003)

compared with b-C which undergoes continuous degradationin leaves during illumination (Beisel et al. 2010). Based on theextent of Phy accumulation in NF-treated Capsicum leaves,Simkin et al. (2003) have estimated a de novo carotenoid syn-thesis rate as high as 50% d–1 of the total carotenoid content inleaves. If the differences in 14C signal levels of b-C + Phy be-tween water-treated leaves and NF-treated leaves at 0 h weredue to such rapid degradation of 14C-labeled b-C upon synthe-sis, as opposed to high stability (or zero degradation) of14C-labeled Phy, about 60–70% of the newly synthesized b-Cmust have been degraded instantly during the 30 min 14CO2

application prior to 0 h. Although such futile synthesis withimmediate degradation cannot be excluded, we think it ismore likely that up-regulation of biosynthesis also played arole in increasing carotenoid labeling in our NF-treatedArabidopsis leaves.

The enhanced 14C labeling of Phy already at 0 h was followedby a further increase at 6 h in the NF-treated leaves under theCL!HL conditions (Fig. 4F). This second increase was accom-panied by a significant decrease in both b-C content (Fig. 3I)and Fv/Fm (Fig. 1C). Neither a b-C decrease alone, as inNF-treated leaves in CL, nor a b-C decrease combined withsevere photoinhibition, as in Linco-treated leaves in CL!HL,led to a further increase in the radioactivity of Phy (or b-C) atthe end of the measurement (6 h; Fig. 4C–F). In the case ofLinco-treated leaves, the 14C signal of b-C even declined.Whatever the mechanisms for the rapid (within 1 h) and slow(�6 h) increase of 14C-labeled b-C + Phy in NF-treated leaves,the total b-C content did decrease in these samples (Figs. 2I,3I), suggesting that b-C degradation could not be stopped (orstrongly down-regulated) to counterbalance short supply.

The substantial loss of b-C was accompanied by a muchreduced increase in V + A + Z in NF-treated leaves comparedwith the control under CL!HL conditions (Fig. 3H and B,respectively). Thus, inhibition of Phy desaturase by NF canlargely suppress the HL-induced increase of V + A + Z inArabidopsis leaves, as has also been observed in duckweed(Garcıa-Plazaola et al. 2002). The residual increase inV + A + Z found in NF-treated leaves may be attributable toincomplete effects of the inhibitor in our experiment, or hy-droxylation of b-C molecules released from photoinactivatedPSII during D1 repair (Depka et al. 1998). However, we wereunable to detect 14C-labeled Z (data not shown), even in thewater-treated or Linco-treated samples in which Z as well asV + A + Z levels were significantly increased under CL!HL(Fig. 3B, E). Altering the length and timing of 14CO2 applicationby, for example, an additional second pulse at a later time or alonger chase of >24 h did not lead to obvious 14C labeling ofZ (or other xanthophylls) in the Arabidopsis leaves. Based onthese observations and considering the presumably minor roleof the b-C hydroxylation during operation of the D1 repair cyclein Arabidopsis plants, it can be hypothesized that HL-inducedsynthesis and accumulation of V + A + Z may utilize a precur-sor pool which is not immediately linked to photosyntheticCO2 fixation. If so, chloroplasts may have two distinct pools

1200 Plant Cell Physiol. 52(7): 1193–1203 (2011) doi:10.1093/pcp/pcr069 ! The Author 2011.

K. G. Beisel et al.

at Forschungszentrum

Juelich Gm

bH, Z

entralbibliothek on July 13, 2011pcp.oxfordjournals.org

Dow

nloaded from

and fluxes of carotenoid precursors in mature Arabidopsisleaves, one for continuous synthesis of b-C in the light (rapidlyderiving carbon from photosynthesis) and another forstress-induced synthesis of V + A + Z and perhaps also Lut(Fig. 3B, C). Further investigations are needed to elucidatethe functional and metabolic interactions between pigmentsand photosynthesis in green leaves.

Conclusion

Both synthesis and degradation in continuous turnover of Chl aseem to be regulated in coordination with D1 protein turnoverin mature leaves of Arabidopsis. The b-C turnover, on the otherhand, is not tightly coupled with Chl a and D1 turnover, pre-sumably because of indirect involvement of b-C in D1 proteinsynthesis and insertion as well as its photoprotective functionrequiring a distinct control. Inhibition of Phy desaturase by NFresults in significantly higher initial labeling of b-C + Phy, sug-gesting up-regulation in metabolic flux down the carotenoidbiosynthetic pathway.

Materials and Methods

Plant materials and growth conditions

Arabdiopsis Columbia-0 wild-type plants were grown in soil(ED 73 Einheitserde, Balster Einheitserdewerk) in a growth cab-inet with a 12 h/12 h day/night photoperiod under constantrelative air humidity of 50% and 22�C/18�C (day/night) airtemperature. A photosynthetically active photon flux densityof approximately 130 mmol photons m–2 s–1 (CL) was providedby a combination of FLUORA and warm white fluorescenttubes (Osram). At the beginning and at the end of the dayperiod, the light intensity in the growth cabinet was graduallyincreased or decreased over 1 h to simulate sunrise and sunset.Mature leaves (up to three leaves per plant) of 6- to 7-weeks-oldplants were used for all experiments.

Inhibitor and light treatments

At the end of the night period, leaves were excised from plantsand the petioles immediately put in water (Control), 3 mMLinco or 70 mM NF solution. Leaves were left to transpire for2 h in the dark under ventilation. During the subsequent lighttreatment for experiments of Chl a fluorescence measure-ments, analysis of photosynthetic pigments and isotope label-ing with 14CO2, leaves were floated on water or diluted inhibitorsolutions (1 mM Linco or 10mM NF) with the adaxial surfacefacing the air. All experiments were conducted under CL(CL!CL condition) or after transfer to HL (1,100 mmol pho-tons m–2 s–1, applied with Master HPI-T Plus lamps; Philips)(CL!HL condition) at an ambient air temperature of 19�C. Apreliminary experiment with fluorescence imaging showed nosignificant spatial variation of Fv/Fm in inhibitor-treated leavesunder the CL!HL condition, suggesting homogeneous effectsof the inhibitors across the lamina.

Chl a fluorescence measurements

Following different durations of exposure to CL!CL orCL!HL conditions, the detached leaves were placed on amoist tissue and dark-adapted for 15 min by using leaf clips.The maximal PSII efficiency, Fv/Fm (fluorescence nomenclatureaccording to van Kooten and Snel 1990), was determined bymeasuring Chl a fluorescence in dark-adapted leaves with aHandy PEA (Hansatech).

Analysis of photosynthetic pigments

For pigment analysis, leaf discs (1.54 cm2) were taken from thedetached leaves at different times during the CL!CL orCL!HL treatment. The discs were frozen in liquid nitrogenand stored at –20�C for up to 2 weeks until acetone extraction.Pigment extraction and the HPLC analysis were performed ac-cording to the method described by Matsubara et al. (2005)using an Allsphere ODS-1 C18 column (5 mm, 250� 4.6 mm;Alltech) and a corresponding guard column (5 mm,7.5� 4.6 mm; Alltech). Pigments were detected by a PAD-996UV/VIS detector (Waters) and peak areas were integrated at440 nm with Waters Empower software. The Chl content wascalculated per unit of leaf area (mmol m–2) and the contents ofdifferent carotenoids were expressed relative to the amount ofChl a (mmol mol–1 Chl a) in each sample.

Isotope labeling with 14CO2 and radio-HPLCanalysis of 14C-labeled pigments14CO2 labeling of leaves was performed in a gas circuit system aspreviously described (Beisel et al. 2010). Detached leaves weresupplied with water or diluted inhibitor solutions (1 mM Lincoor 10mM NF) during administration of 14CO2 released by acid-ification of aqueous sodium [14C]carbonate (1.85 MBq per leaf;GE Healthcare) under CL conditions at 19�C. Immediately aftera 30 min application of 14CO2, some leaves were harvested foranalysis (time 0 h) while others were floated on water or dilutedinhibitor solutions in ambient air under CL!CL or CL!HLconditions for different chase periods before harvesting forradio-HPLC analysis. The radiolabeled pigments were extractedfirst with 1.2 ml of acetone, followed by two-phase extraction(ethyl acetate and water) performed twice according to themethod of Pogson et al. (1996). The extracts were concentratedto a final volume of 200 ml under a nitrogen gas stream and dimlaboratory light. The concentrated extracts were either imme-diately analyzed by radio-HPLC (100ml injection volume) orstored at –20�C for <5 h until analysis. Radio-HPLC analysiswas carried out with a Prontosil reversed-phase C30 column(3 mm, 250� 4.6 mm; Bischoff ) and a corresponding guard cart-ridge (3 mm, 10� 4.0 mm; Bischoff ) according to Beisel et al.(2010). The pigments were detected with a UV/VIS detector(Jasco) and radioactivity by a Radioflow detector LB 509(Berthold Technologies) with a time delay of 20 s betweenthe two detectors (Fig. 5).

Peak integration was performed with RadioStar software(Berthold Technologies) for both UV/VIS chromatograms

1201Plant Cell Physiol. 52(7): 1193–1203 (2011) doi:10.1093/pcp/pcr069 ! The Author 2011.

Turnover of b-carotene and Chl a in leaves

at Forschungszentrum

Juelich Gm

bH, Z

entralbibliothek on July 13, 2011pcp.oxfordjournals.org

Dow

nloaded from

(286 and 440 nm for Phy and photosynthetic pigments, respect-ively) and radiograms. Peak areas of the radiogram were nor-malized to the Chl a content obtained from the corresponding440 nm chromatogram and expressed as Bq mg–1 Chl a.

An extra peak found in the radiogram of NF-treated leaves(Fig. 5C) was identified as Z-phytoene (Phy) by adding a purestandard of (E/Z)-phytoene (CaroteNature) to a leaf extractand measuring the absorbance at 286 nm (Fig. 5B).

Statistical data analysis

Pigment contents and 14C labeling data were statisticallytested by one-way analysis of variance (ANOVA; Dunnett’smethod). Pigment contents were checked for significant differ-ences in time-course variations within each treatment bycomparing data at 0.5, 3 and 6 h with 0 h. Differences in 14Cincorporation between inhibitor-treated and water-treated(Control) leaves were statistically tested at each samplingtime point.

Acknowledgments

We thank Diana Hofmann and Stephan Koppchen(Forschungszentrum Julich) for valuable suggestions and tech-nical assistance with radio-HPLC analysis. Critical comments ofSiegfried Jahnke (Forschungszentrum Julich) and Ralf Welsch(University of Freiburg) on the early version of the manuscriptare greatly appreciated. K.G.B. acknowledges the support of herPh.D. thesis at the Heinrich-Heine-Universitat Dusseldorf.

References

Auldridge, M.E., McCarty, D.R. and Klee, H.J. (2006) Plant carotenoidcleavage oxygenases and their apocarotenoid products. Curr. Opin.Plant Biol. 9: 315–321.

Baena-Gonzalez, E. and Aro, E.-M. (2002) Biogenesis, assembly andturnover of photosystem II units. Philos. Trans. R. Soc. B: Biol. Sci.357: 1451–1459.

Bautista, J.A., Rappaport, F., Guergova-Kuras, M., Cohen, R.O.,Golbeck, J.H., Wang, J.Y. et al. (2005) Biochemical and biophysicalcharacterization of photosystem I from phytoene desaturase andz-carotene desaturase deletion mutants of Synechocystis sp PCC6803. J. Biol. Chem. 280: 20030–20041.

Beisel, K.G., Jahnke, S., Hofmann, D., Koppchen, S., Schurr, U. andMatsubara, S. (2010) Continuous turnover of carotenes and chloro-phyll a in mature leaves of Arabidopsis revealed by 14CO2 pulse–chase labeling. Plant Physiol. 152: 2188–2199.

Breitenbach, J., Zhu, C. and Sandmann, G. (2001) Bleaching herbicidenorflurazon inhibits phytoene desaturase by competition with thecofactors. J. Agric. Food Chem. 49: 5270–5272.

Cazzonelli, C.I. and Pogson, B.J. (2010) Source to sink: regulation ofcarotenoid biosynthesis in plants. Trends Plant Sci. 15: 266–274.

Corona, V., Aracri, B., Kosturkova, G., Bartley, G.E., Pitto, L., Giorgetti, L.et al. (1996) Regulation of a carotenoid biosynthesis gene promoterduring plant development. Plant J. 9: 505–512.

Cuttriss, A.J., Chubb, A.C., Alawady, A., Grimm, B. and Pogson, B.J.(2007) Regulation of lutein biosynthesis and prolamellar body for-mation in Arabidopsis. Funct. Plant Biol. 34: 663–672.

Dalla Vecchia, F., Barbato, R., La Rocca, N., Moro, I. and Rascio, N.(2001) Responses to bleaching herbicides by leaf chloroplasts ofmaize plants grown at different temperatures. J. Exp. Bot. 52:811–820.

Demmig-Adams, B. (1990) Carotenoids and photoprotection inplants: a role for the xanthophyll zeaxanthin. Biochim. Biophys.Acta 1020: 1–24.

Demmig-Adams, B. and Adams, W.W. III (1992) Carotenoid compos-ition in sun and shade leaves of plants with different life forms.Plant Cell Environ. 15: 411–419.

Depka, B., Jahns, P. and Trebst, A. (1998) b-Carotene to zeaxanthinconversion in the rapid turnover of the D1 protein of photosystemII. FEBS Lett. 424: 267–270.

Fiekers, J.F., Marshall, I.G. and Parsons, R.L. (1979) Clindamycin andlincomycin alter miniature endplate current decay. Nature 281:680–682.

Forster, B., Osmond, C.B. and Pogson, B.J. (2009) De novo synthesisand degradation of Lx and V cycle pigments during shade and sunacclimation in avocado leaves. Plant Physiol. 149: 1179–1195.

Garcıa-Plazaola, J.I., Hernandez, A., Artetxe, U. and Becerril, J.M. (2002)Regulation of the xanthophyll cycle pool size in duckweed (Lemnaminor) plants. Physiol. Plant. 116: 121–126.

Havaux, M. and Niyogi, K. (1999) The violaxanthin cycle protectsplants from photooxidative damage by more than one mechanism.Proc. Natl Acad. Sci. USA 96: 8762–8767.

He, Q.F. and Vermaas, W. (1998) Chlorophyll a availability affects psbAtranslation and D1 precursor processing in vivo in Synechocystis sp.PCC 6803. Proc. Natl Acad. Sci. USA 95: 5830–5835.

Jahns, P., Latowski, D. and Strzal�ka, K. (2009) Mechanism and regula-tion of the violaxanthin cycle: the role of antenna proteins andmembrane lipids. Biochim. Biophys. Acta 1787: 3–14.

Keren, N., Berg, A., Van Kan, P.J.M., Levanon, H. and Ohad, I. (1997)Mechanism of photosystem II photoinactivation and D1 proteindegradation at low light: the role of back electron flow. Proc. NatlAcad. Sci. USA 94: 1579–1584.

Kim, J., Eichacker, L.A., Rudiger, W. and Mullet, J.E. (1994) Chlorophyllregulates accumulation of the plastid-encoded chlorophyll proteinsP700 and D1 by increasing apoprotein stability. Plant Physiol. 104:907–916.

Kim, J., Smith, J.J., Tian, L. and DellaPenna, D. (2009) The evolution andfunction of carotenoid hydroxylases in Arabidopsis. Plant CellPhysiol. 50: 463–479.

Kobayashi, M., Maeda, H., Watanabe, T., Nakane, H. and Satoh, K.(1990) Chlorophyll a and b-carotene content in the D1/D2/cyto-chrome b559 reaction center complex from spinach. FEBS Lett. 260:138–140.

Lee, H.Y., Hong, Y.N. and Chow, W.S. (2001) Photoinactivationof photosystem II complexes and photoprotection by non-functional neighbours in Capsicum annuum L. leaves. Planta 212:332–342.

Maeda, H. and DellaPenna, D. (2007) Tocopherol functions in photo-synthetic organisms. Curr. Opin. Plant Biol. 10: 260–265.

Markgraf, T. and Oelmuller, R. (1991) Evidence that carotenoids arerequired for the accumulation of a functional photosystem II, butnot photosystem I in the cotyledons of mustard seedlings. Planta185: 97–104.

1202 Plant Cell Physiol. 52(7): 1193–1203 (2011) doi:10.1093/pcp/pcr069 ! The Author 2011.

K. G. Beisel et al.

at Forschungszentrum

Juelich Gm

bH, Z

entralbibliothek on July 13, 2011pcp.oxfordjournals.org

Dow

nloaded from

Matsubara, S. and Chow, W.S. (2004) Populations of photoinactivatedphotosystem II reaction centers characterized by chlorophyll afluorescence lifetime in vivo. Proc. Natl Acad. Sci. USA 101:18234–18239.

Matsubara, S., Krause, G.H., Aranda, J., Virgo, A., Beisel, K.G., Jahns, P.et al. (2009) Sun–shade patterns of leaf carotenoid composition in86 species of neotropical forest plants. Funct. Plant Biol. 36: 20–36.

Matsubara, S., Morosinotto, T., Bassi, R., Christian, A.L., Fischer-Schliebs, E., Luttge, U. et al. (2003) Occurrence of the lutein-epoxidecycle in mistletoes of the Loranthaceae and Viscaceae. Planta 217:868–879.

Matsubara, S., Naumann, M., Martin, R., Nichol, C., Rascher, U.,Morosinotto, T. et al. (2005) Slowly reversible de-epoxidation oflutein-epoxide in deep shade leaves of a tropical tree legume may‘lock-in’ lutein-based photoprotection during acclimation to stronglight. J. Exp. Bot. 56: 461–468.

Melis, A. (1999) Photosystem II damage and repair cycle in chloro-plasts: what modulates the rate of photodamage in vivo? TrendsPlant Sci. 4: 130–135.

Meskauskiene, R., Nater, M., Goslings, D., Kessler, F., den Camp, R.O.and Apel, K. (2001) FLU: a negative regulator of chlorophyll biosyn-thesis in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 98:12826–12831.

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

Noctor, G. and Foyer, C.H. (1998) Ascorbate and glutathione: keepingactive oxygen under control. Annu. Rev. Plant Physiol. PlantMol. Biol. 49: 249–279.

Norris, S.R., Barrette, T.R. and DellaPenna, D. (1995) Genetic dissectionof carotenoid synthesis in arabidopsis defines plastoquinone as anessential component of phytoene desaturation. Plant Cell 7:2139–2149.

Rossel, J.B., Wilson, I.W. and Pogson, B.J. (2002) Global changes in geneexpression in response to high light in Arabidopsis. Plant Physiol.130: 1109–1120.

Sandmann, G., Kuhn, M. and Boger, P. (1993) Carotenoids in photo-synthesis—protection of D1 degradation in the light. Photosynth.Res. 35: 185–190.

Simkin, A.J., Zhu, C., Kuntz, M. and Sandmann, G. (2003) Light–darkregulation of carotenoid biosynthesis in pepper (Capsicumannuum) leaves. J. Plant Physiol. 160: 439–443.

Sozer, O., Komenda, J., Ughy, B., Domonkos, I., Laczko-Dobos, H.,Malec, P. et al. (2010) Involvement of carotenoids in the synthesis

and assembly of protein subunits of photosynthetic reactioncenters of Synechosystis sp. PCC 6803. Plant Cell Physiol. 51:823–835.

Telfer, A. (2005) Too much light? How b-carotene protects thephotosystem II reaction centre. Photochem. Photobiol. Sci. 4:950–956.

Tracewell, C.A., Vrettos, J.S., Bautista, J.A., Frank, H.A. andBrudvig, G.W. (2001) Carotenoid photooxidation in photosystemII. Arch. Biochem. Biophys. 385: 61–69.

Trebst, A. and Depka, B. (1997) Role of carotene in the rapid turnoverand assembly of photosystem II in Chlamydomonas reinhardtii.FEBS Lett. 400: 359–362.

Tyystjarvi, E., Aliyrkko, K., Kettunen, R. and Aro, E.-M. (1992) Slowdegradation of the D1 protein is related to the susceptibility oflow-light-grown pumpkin plants to photoinhibition. Plant Physiol.100: 1310–1317.

Tyystjarvi, E. and Aro, E.-M. (1996) The rate constant of photo-inhibition, measured in lincomycin-treated leaves, is directly0proportional to light intensity. Proc. Natl Acad. Sci. USA 93:2213–2218.

van Kooten, O. and Snel, J.F.H. (1990) The use of chlorophyll fluores-cence nomenclature in plant stress physiology. Photosynth. Res. 25:147–150.

Vavilin, D. and Vermaas, W. (2007) Continuous chlorophyll degrad-ation accompanied by chlorophyllide and phytol reutilization forchlorophyll synthesis in Synechocystis sp PCC 6803. Biochim.Biophys. Acta 1767: 920–929.

Verhoeven, A.S., Adams, W.W. III, Demmig-Adams, B., Croce, R. andBassi, R. (1999) Xanthophyll cycle pigment localization and dynam-ics during exposure to low temperatures and light stress in Vincamajor. Plant Physiol. 120: 727–737.

Welsch, R., Medina, J., Giuliano, G., Beyer, P. and von Lintig, J. (2003)Structural and functional characterization of the phytoene synthasepromoter from Arabidopsis thaliana. Planta 216: 523–534.

Xu, C.C., Kuang, T.Y., Li, L.B. and Lee, C.H. (2000) D1 protein turnoverand carotene synthesis in relation to zeaxanthin epoxidation in riceleaves during recovery from low temperature photoinhibition. Aust.J. Plant Physiol. 27: 239–244.

Zhang, L.X., Paakkarinen, V., van Wijk, K.J. and Aro, E.-M. (1999)Co-translational assembly of the D1 protein into photosystem II.J. Biol. Chem. 274: 16062–16067.

1203Plant Cell Physiol. 52(7): 1193–1203 (2011) doi:10.1093/pcp/pcr069 ! The Author 2011.

Turnover of b-carotene and Chl a in leaves

at Forschungszentrum

Juelich Gm

bH, Z

entralbibliothek on July 13, 2011pcp.oxfordjournals.org

Dow

nloaded from