ORIGINAL ARTICLE

Occurrence of the lutein-epoxide cycle in mistletoesof the Loranthaceae and Viscaceae

Received: 11 March 2003 / Accepted: 24 May 2003 / Published online: 3 July 2003� Springer-Verlag 2003

Abstract The lutein-epoxide cycle (Lx cycle) is anauxiliary xanthophyll cycle known to operate only insome higher-plant species. It occurs in parallel with thecommon violaxanthin cycle (V cycle) and involvesthe same epoxidation and de-epoxidation reactions as inthe V cycle. In this study, the occurrence of the Lx cyclewas investigated in the two major families of mistletoe,the Loranthaceae and the Viscaceae. In an attempt tofind the limiting factor(s) for the occurrence of the Lxcycle, pigment profiles of mistletoes with and withoutthe Lx cycle were compared. The availability of luteinas a substrate for the zeaxanthin epoxidase appeared notto be critical. This was supported by the absence of theLx cycle in the transgenic Arabidopsis plant lutOE, inwhich synthesis of lutein was increased at the expense of

V by overexpression of e-cyclase, a key enzyme for luteinsynthesis. Furthermore, analysis of pigment distributionwithin the mistletoe thylakoids excluded the possibilityof different localizations for the Lx- and V-cycle pig-ments. From these findings, together with previous re-ports on the substrate specificity of the two enzymes inthe V cycle, we propose that mutation to zeaxanthinepoxidase could have resulted in altered regulation and/or substrate specificity of the enzyme that gave rise tothe parallel operation of two xanthophyll cycles in someplants. The distribution pattern of Lx in the mistletoephylogeny inferred from 18S rRNA gene sequences alsosuggested that the occurrence of the Lx cycle is deter-mined genetically. Possible molecular evolutionary pro-cesses that may have led to the operation of the Lx cyclein some mistletoes are discussed.

Keywords Carotenoid Æ Loranthaceae Æ Luteinepoxide Æ Viscaceae Æ Xanthophyll cycle Æ Zeaxanthinepoxidase

Abbreviations A: antheraxanthin Æ a- and b-Car: a- andb-carotene Æ Chl: chlorophyll Æ a-DM: dodecyl-a-D-maltoside Æ DPS: de-epoxidation state of the violax-anthin cycle (= [A+Z]/[V+A+Z]) Æ Lut: lutein Æ Lx:lutein epoxide Æ S Caro: total carotenoid concent-ration Æ V: violaxanthin Æ VAZ: pool size of theviolaxanthin cycle (= V+A+Z) Æ VDE: violaxanthinde-epoxidase Æ Z: zeaxanthin Æ ZE: zeaxanthin epoxidase

Introduction

Since the advent of photosynthesis, photoautotrophshave evolved a wide variety of carotenoids along withchlorophylls (Chl) to utilize light energy efficiently. Inthe case of higher plants, only six different carotenoidstypically accumulate and additions are uncommon(Young 1993a). They contribute to light harvesting on

Planta (2003) 217: 868–879DOI 10.1007/s00425-003-1059-7

Shizue Matsubara Æ Tomas Morosinotto Æ Roberto Bassi

Anna-Luise Christian Æ Elke Fischer-Schliebs

Ulrich Luttge Æ Birgit Orthen Æ Augusto C. Franco

Fabio R. Scarano Æ Britta Forster Æ Barry J. Pogson

C. Barry Osmond

S. Matsubara (&) Æ B. Forster Æ B. J. PogsonResearch School of Biological Sciences,and School of Biochemistry and Molecular Biology,Australian National University,GPO Box 475, ACT 2601 Canberra, AustraliaE-mail: matsubara@rsbs.anu.edu.auFax: +61-2-61258056

T. Morosinotto Æ R. BassiDipartimento Scientifico e Tecnologico,Universita di Verona, Strada Le Grazie,15-37234 Verona, Italy

A.-L. Christian Æ E. Fischer-Schliebs Æ U. LuttgeInstitut fur Botanik, Technische Universitat Darmstadt,Schnittspahnstrasse 3–5, 64287 Darmstadt, Germany

B. Orthen Æ A. C. FrancoDepartamento de Botanica, Universidade de Brasılia,caixa postal 04457, 70919-970 Brasılia, DF, Brazil

F. R. ScaranoDepartamento de Ecologia, Universidade Federal do Rio deJaneiro, CCS, Ilha do Fundao, 21941-970 Rio de Janeiro, RJ,Brazil

C. B. OsmondBiosphere 2 Center, Columbia University,Oracle, AZ 85623, USA

one hand, ensuring the driving force of photosynthesis,and to photoprotection on the other, preventing damageto the photosynthetic machinery when the light becomesexcessive and hence harmful.

Among many different carotenoids, three pigments inthe xanthophyll cycle (Fig. 1), i.e. violaxanthin (V), an-theraxanthin (A), and zeaxanthin (Z), are thought toplay a key role in photoprotective thermal energy dis-sipation (reviewed by Demmig-Adams 1990; Yamamotoand Bassi 1996; Gilmore and Govindjee 1999; Niyogi1999). Under illumination, V, which is supposedlyinactive in energy dissipation, is converted to the activeforms, Z and A, by the enzyme violaxanthin de-epoxi-dase (VDE; Yamamoto et al. 1962). Reverse reactions,which are catalysed by zeaxanthin epoxidase (ZE),become noticeable in the absence of VDE activity atneutral pH (Siefermann and Yamamoto 1975), i.e. in thedark or under low light in natural environments. Theoperation of the violaxanthin cycle (or V cycle) seems tobe a common feature of higher plants and has indeedbeen observed in all species thus far investigated.

Recently, operation of another xanthophyll cyclehas been documented in two parasitic plants, Cuscutareflexa Roxb. (Bungard et al. 1999) and Amyema mi-quelii (Lehm. ex Miq.) Tiegh. (Matsubara et al. 2001).This auxiliary xanthophyll cycle (Fig. 1), originallydiscovered in green tomato fruit by Rabinowitch et al.(1975), operates in parallel with the V cycle with one-step interconversions between lutein epoxide (Lx) andlutein (Lut). It seems that the two xanthophyll cyclesare regulated by similar mechanisms except that epox-idation reactions are somewhat slower in the Lx–Lutcycle (or Lx cycle) compared to the V cycle (Matsu-bara et al. 2001; Garcıa-Plazaola et al. 2003). Fur-thermore, the Lx cycle differs from the V cycle in thatit occurs in the a-carotene (a-Car) branch of thecarotenoid biosynthesis pathway whereas the V cycle

operates in the b-carotene (b-Car) branch (Fig. 1).Although the operation of the Lx cycle has beenobserved in different oak trees as well (Garcıa-Plazaolaet al. 2002), it is apparently not widespread amonghigher plant species. The factors and mechanisms thatdetermine the occurrence (or absence) of the Lx cycleas well as its physiological significance are yet to beelucidated.

The finding of the Lx cycle in the Australian mistletoeA. miquelii raised the question of whether it is also to befound in various taxa of mistletoe plants. Being thelargest and most diverse group of aerial parasites, mis-tletoes represent about half of all parasitic angiosperms(Press and Gurney 2000). The family Loranthaceae,which is one of the two major families of mistletoes andto which A. miquelii belongs, comprises ca. 950 species in65 genera (Calder 1997). Based on the essentiallysouthern and pantropical distribution, it has beenassumed that the Loranthaceae has its origin inGondwanaland. On the contrary, the Viscaceae, theother major group with ca. 400 species in 7 generaincluding the European Christmas mistletoe Viscumalbum L., is well distributed in both hemispheres. Thoughthe two families are both mistletoes, they are supposed tohave evolved independently (Kuijt 1969; Barlow 1983). Ifthe occurrence of the Lx cycle is associated with muta-tion to the operational component(s) of the V cycle, asurvey on the Lx distribution in these two mistletoefamilies could reveal the Lx-cycle phylogeny.

In the present study, we therefore investigated thepigment composition in mistletoe species of the familiesLoranthaceae and Viscaceae to address the questionswhat could be a controlling step for the Lx cycle to takeplace and whether the distribution pattern of the Lx cyclematches the mistletoe phylogeny.

Materials and methods

Plant material

Plant species used for pigment analysis and their origins are listedin Table 1. Samples from Australian species were collected inFebruary [Amyema (A.)cambagei and Exocarpos (E.) cupressifor-mis] and October 2000 (A. miquelii), April (A. pendulum) andDecember 2001 [Muellerina (M.) eucalyptoides], and December2002 [Nuytsia (N.) floribunda]. European species were collected inthe Darmstadt Botanic Garden [Viscum (V.) album, Arceuthobium(Ar.) oxycedri, and Melampyrum (Me.) pratense] and in the sur-roundings (V. laxum) in May 2002. Species of North and SouthAmerica were collected in Arizona and Brazil in August and Sep-tember 2002, respectively. All samples except for Ar. divarica-tumand Phoradendron (P.) juniperinum were dark-adapted for>2 h at room temperature before freezing in liquid nitrogen.Samples of Ar. divaricatum and P. juniperinum were frozen in liquidnitrogen on the site of collection without dark-adaptation. For Ar.divaricatum, we used only female plants to avoid contaminationwith floral pigments, which was inevitable for male plants under thesampling condition in the field. Frozen materials were then storedeither at )70 �C or in liquid nitrogen until pigment analysis.

The transgenic plant of Arabidopsis thaliana, lutOE, was gen-erated as described in Pogson and Rissler (2000). In lutOE, luteincontent was substantially increased at the expense of V by

Fig. 1 Reactions in the violaxanthin cycle (right) and the lutein-epoxide cycle (left) operating in the carotenoid biosynthesispathway of plants

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overexpression of e-cyclase (Fig. 1), a limiting enzyme of luteinsynthesis (Pogson et al. 1996). Plants were grown under light/darkcycle of 18 h/6 h (100 lmol photons m)2 s)1) at 21 �C.

For the analysis of pigment distribution within the thylakoidmembranes, a V. album plant was collected in Moutiers (France) inApril 2002. Thylakoid membranes were isolated from dark-adaptedleaves, solubilized, and fractionated as described below.

High light treatment

In order to test the light-dependent conversion of V and Lx,leaves of A. pendulum, Phthirusa (Phth.) ovata, and V. album weresubjected to high light treatment. Leaf samples were dark-adaptedat room temperature for >2 h prior to the light treatment. For A.miquelii and Phth. ovata, two discs were removed from each leafafter dark-adaptation and one disc was frozen immediately inliquid nitrogen while the other disc was treated by high lightbefore freezing. For V. album, dark and high light samples weretaken from different leaves of a plant. The high light treatmentwas at 1,700 lmol photons m)2 s)1 at 23 �C for 2 h for A. pen-dulum and at 800 lmol photons m)2s)1 at 28 �C for 1.5 h forPhth. ovata and V. album.

Preparation and solubilization of thylakoids

Dark-adapted leaves of V. album were sliced into ca. 0.5-mm-thickpieces on an ice-cold plate under dim light. Leaf pieces were thenincubated in a solution containing 0.6 M sorbitol, 20 mM Mes(pH 5.5), 50 mM ascorbate, 5 mM MgCl2, 0.2% BSA, 2% cellu-lase, 0.1% macerozyme (Bassi and Simpson 1986), and proteaseinhibitors (0.2 mM benzamidin, 1 mM e-amino-n-caproic acid,and 0.2 mM phenylmethylsulfonyl fluoride) under gentle agitationat 30 �C for 2.5 h in the dark. This enzymatic digestion wasnecessary to facilitate proper isolation of thylakoids from mistle-toe leaves.

Samples were then homogenized in T1 solution containing0.1 M Tricine (pH 7.8), 0.4 M sorbitol, 0.5% milk powder, and thesame set of protease inhibitors as in the digestion solution. Theslurry was filtered through one layer of 20-lm nylon mesh and thefiltrate centrifuged at 1,400 g at 4 �C for 10 min. The thylakoidpellet was resuspended in T1 solution three more times to removethe added enzymes as well as proteins from the cytoplasm. Thewashed pellet was twice resuspended in T2 solution containing25 mM Hepes (pH 7.5) and 10 mM EDTA and centrifuged at10,000 g at 4 �C for 15 min. The obtained thylakoids were resus-pended in T3 solution containing 50% (v/v) glycerol, 10 mMHepes(pH 7.5), and 1 mM EDTA, and kept frozen until use.

Table 1 List of plants used for the pigment analyses

Order Family Genus Species Authority Origin Comments

Santalales Loranthaceae Amyema (A.) cambagei (Blakely) Danser Canberra, ACT, Australia –miquelii (Lehm. ex Miq.) Tiegh. Canberra, ACT, Australia –pendulum (Sieb. ex Spreng.) Tiegh. Canberra, ACT, Australia Needle-like

leavesMuellerina (M.) eucalyptoides (DC.) Barlow Canberra, ACT, Australia –Nuytsia (N.) floribunda (Labill.) R. Br. Perth, WA, Australia Root-parasitic

mistletoePhthirusa (Phth.) ovata Eichl. Brasilia, DF, Brazil –Psittachanthus (Ps.) dichrous Mart. Arraial do Cabo, RJ,

Brazil–

robustus Mart. Brasilia, DF, Brazil –Struthanthus (S.) flexicaulis Mart. Brasilia, DF, Brazil –

marginatus (Desr.) Bl. Macae, RJ, Brazil –Viscaceae Arceuthobium (Ar.) divaricatum Engelm. Grand Canyon, AZ,

USADwarf mistletoe

oxycedri (DC.) M. Bieb. Darmstadt BotanicGarden, Germany

Dwarf mistletoe

Phoradendron (P.) californicum Nutt. Oracle, AZ, USA Nearly leaflessdipterum Eichl. Brasilia, DF, Brazil –emarginatum Mart. ex Eichl. Brasilia, DF, Brazil –hexastichon Griseb. Brasilia, DF, Brazil –juniperinum Engelm. ex Gray Grand Canyon, AZ,

USANearly leafless

perrottetii Nutt. Brasilia, DF, Brazil –piauhyanum Trel. Brasilia, DF, Brazil –piperoides Trel. Brasilia, DF, Brazil –semivenosum Rizz. Brasilia, DF, Brazil –tunaeforme Eichl. Brasilia, DF, Brazil Nearly leaflessundulatum Eichl. Brasilia, DF, Brazil –

Viscum (V.) album L. Darmstadt BotanicGarden, Germany

–

laxum Boiss. & Reuter Muhltal-Trautheim,Hessen, Germany

–

Santalaceae Exocarpos (E.) cupressiformis Labill. Canberra, ACT,Australia

Root parasite

Lamiales Orobanchaceae Melampyrum (Me.) pratense L. Darmstadt BotanicGarden, Germany

Root parasite

Laurales Lauraceae Cassytha (Ca.) filiformis L. Arraial do Cabo,RJ, Brazil

Dodder-likeparasite

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Thylakoid samples were washed with 5 mM EDTA prior tosolubilization with 1% dodecyl-a-D-maltoside (a-DM) in 10 mMHepes (pH 7.5). Because mistletoe thylakoids were found to bevery difficult to solubilize with a mild detergent such as a-DM, weincubated the thylakoids in the solubilization buffer on ice undergentle agitation in the dark until they were fully solubilized (for0.5–1 h).

Sucrose gradient ultracentrifugation

Solubilized membranes were fractionated by ultracentrifugation ina 0.1–1 M sucrose gradient containing 10 mM Hepes (pH 7.5) and0.06% a-DM at 280,000 g at 4 �C for 20 h. Five bands and a pelletwere isolated from the gradient tubes. The pellet collected from thebottom of the tubes was further purified by another sucrose gra-dient. The polypeptide composition of each fraction was identifiedby SDS–PAGE (14% acrylamide, Tris/Tricine buffer system),room-temperature absorption spectra (DW-2000 spectrophotome-ter; SLM-Aminco, Silver Spring, MD, USA), and pigment analysis(see below) as described previously (Dainese and Bassi 1991;Caffarri et al. 2001).

Pigment assay

Pigments were extracted from frozen samples as described inMatsubara et al. (2001) and analysed by HPLC. The HPLCmethod used for A. miquelii, M. eucalyptoides, N. floribunda, andthe Arabidopsis mutant was according to Pogson et al. (1996). Forall other samples including thylakoid membrane fractions from V.album, the method by Gilmore and Yamamoto (1991) was em-ployed. In addition, the HPLC analysis results for the thylakoidfractions were verified by fitting the absorption spectra of thesample extracts with the spectra of pure pigments (Connelly et al.1997).

Phylogenetic analysis

Phylogenetic trees were constructed based on 18S rRNA gene se-quences of mistletoe species available from GenBank. At least onespecies for each mistletoe genus investigated in the pigment survey,except forMuellerina and Nuytsia, was included in the phylogeneticanalysis, albeit not always the same species. GenBank accessionnumbers of the 18S rRNA gene sequences used in this study are:AF039073 (A. glabrum), L24412 (Phth. pyrifolia), L24414 [Psitt-acanthus (Ps.) angustifolius], L24421 [Struthanthus (S.) oerstedii],

L24426 (V. album), L24427 (V. articulatum), L24081 (Ar. oxycedri),L24082 (Ar. pendens), L24042 (Ar. verticilliflorum), AF039070 (P.californicum), X16607 (P. serotinum), and L24142 (E. bidwillii).

Phylogenetic analysis was performed using the neighbor join-ing method. The maximum likelihood method resulted in identicaltree topology with E. bidwillii as the outgroup (data not shown).The same topology was obtained by using different distancemeasures (p distances, Jukes-Cantor, and Kimura 2-parameter).Bootstrap analysis using 10,000 bootstrap replicates was con-ducted to assess the statistical confidence of the branching. Thesoftwares used for the analyses are available from RibosomalDatabase Project II (Maidak et al. 2001) at http://rdp.cme.m-su.edu and from Data Analysis in Molecular Biology and Evo-lution (Xia and Xie 2001) at http://aix1.uottawa.ca/�xxia/software/software.htm.

Results

Carotenoid composition in mistletoe speciesof the Loranthaceae

The carotenoid concentrations in 10 mistletoe species ofthe family Loranthaceae (Table 1) are summarized inTable 2. Neoxanthin (Neo) concentrations ranged be-tween 30 and 50 mmol on a Chl basis (mmolmol)1Chl), which was comparable to the values re-ported for many different sun and shade plants (Thayerand Bjorkman 1990; Demmig-Adams and Adams 1992;Demmig-Adams 1998). The levels of the three V-cyclepigments (V, A, and Z) and the pool size of the V cycle(VAZ) varied substantially between samples, probablyreflecting different growth light environments and/orconditions on the day of sampling. Despite the pre-treatment in the dark, some species [A. pendulum, Ps.)dichrous, and Ps. robustus] still contained considerableamounts of de-epoxidized xanthophylls (A and Z),resulting in high de-epoxidation state values,DPS=(A+Z)/(V+A+Z). This symptom has beenobserved in many plants under stress conditions(Adams et al. 1995; Barker et al. 2002), including mis-tletoes during winter seasons (Matsubara et al. 2002).

Table 2 Carotenoid concentrations in mistletoe species of thefamily Loranthaceae. The values are means of three to six samples± SD (mmol/mol Chl a+b). Neo Neoxanthin, V violaxanthin,A antheraxanthin, Z zeaxanthin, VAZ the pool size of the viola-

xanthin cycle (V+A+Z), DPS de-epoxidation state of the viola-xanthin cycle (A+Z)/(V+A+Z), Lx lutein epoxide, Lut lutein,b-Car b-carotene, S Caro total carotenoid concentration

Genus Species Neo V A Z VAZ DPS Lx Lut Lx/V b-Car S Caro

Amyema miquelii 32±3 54±7 4±0 nda 58±7 0.1±0.0 43±5 105±8 0.8±0.1 84±19 321±24cambagei 45±6 119±18 3±1 nd 122±19 0.0±0.0 23±2 124±16 0.2±0.0 108±35 421±70pendulum 34±2 93±13 17±3 2±3 111±13 0.2±0.1 18±2 122±12 0.2±0.0 112±20 398±39

Muellerina eucalyptoides 40±1 65±3 4±1 nd 69±4 0.1±0.0 24±1 168±2 0.4±0.0 87±1 387±9Nuytsia floribunda 49±4 43±4 1±1 1±1 45±5 0.0±0.0 30±5 150±7 0.7±0.1 92±3 364±15Phthirusa ovata 40±3 88±25 2±2 nd 91±26 0.0±0.0 10±3 200±31 0.1±0.1 91±14 432±72Psittachanthus dichrous 50±2 87±27 10±6 7±6 103±32 0.2±0.1 8±3 164±19 0.1±0.1 86±3 412±50

robustus 45±2 49±18 49±24 100±5 198±34 0.8±0.1 5±2 278±9 0.1±0.1 138±15 663±27Struthanthus flexicaulis 43±3 77±19 5±2 nd 83±18 0.1±0.0 22±3 172±19 0.3±0.1 85±6 405±35

marginatusb 44±1 89±8 6±1 1±1 96±8 0.1±0.0 13±2 149±7 0.2±0.0 74±3 383±19

aConcentrations below the detection limit are shown as ndbThe species contained a-carotene (7±2)

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Generally, a large VAZ was found in those speciesshowing sustained de-epoxidation of the V cycle.

All the examined species of Loranthaceae accumu-lated Lx. The highest concentration of >40 mmolmol)1Chl was found in A. miquelii, in which the par-allel operation of the Lx and V cycles has previouslybeen demonstrated (Matsubara et al. 2001). Two spe-cies that contained relatively low amounts of Lx,namely Ps. dichrous and Ps. robustus, were those withhigher DPS values, suggesting that Lx was partly de-epoxidized to Lut in these plants. For comparison ofthe pool sizes of the Lx and V cycles, we used the ratioLx/V rather than (Lx+Lut)/(V+A+Z). Unlike theV cycle, the pool size of the Lx cycle cannot be esti-mated by simply adding the concentrations of Lx andLut. It should be remembered that Lut, which is boundin the central position of the light-harvesting antennacomplex, is the most abundant xanthophyll pigment inleaves of many higher plants. The majority of Lut isnot involved in the Lx cycle (Matsubara et al. 2001).The Lx/V values for A. miquelii (0.8±0.1) and theroot-parasitic mistletoe N. floribunda (0.7±0.1) clearlystood out among different Loranthacean species, withthe level of Lx in others ranging between 10 and 40%of V.

b-Car comprised 20–30% of the total carotenoidconcentrations (S Caro) in all Loranthacean species.a-Car, which has often been found in shade-tolerant orshade-grown plants (Thayer and Bjorkman 1990;Demmig-Adams and Adams 1992; Demmig-Adams1998), was detected only in S. marginatus at very lowconcentrations (7±2 mmol mol)1Chl). Ps. robustus, inwhich very high S Caro (>660 mmol mol)1Chl) wasmeasured, was one of the two species that retainednotable amounts of Z and A even after dark-adapta-tion.

Carotenoid composition in mistletoe speciesof the Viscaceae

Pigment assays were also conducted for members of theViscaceae (Table 3). Principally, the levels of Neo andVAZ were comparable to those in the Loranthaceanspecies. Again, the three species that exhibited large-VAZ, i.e. Ar. divaricatum, P. semivenosum, and P. und-ulatum, had concomitantly high DPS values although itshould be noted that the samples of Ar. divaricatum werenot dark-adapted. Sustained de-epoxidation of theV cycle was most pronounced in P. undulatum, in whichthe highest DPS value of 0.8 was observed.

A different pattern emerges for the viscacean speciesin terms of the Lx cycle. The levels of Lx measured inthese plants were either very low or less than a traceamount in spite of the low DPS values for most of thespecies, implying that the V-cycle pigments were mainlyepoxidized. Trace levels of Lx have also been found inother higher plant species (Young 1993b). Although theaverage values of Lx (or Lx/V) were very low for the twoViscum species, we observed that some samples con-tained slightly higher amounts of Lx than the others.The highest Lx concentrations found in the Viscummistletoes were 8.0 mmol mol)1Chl and 8.1 mmolmol)1Chl for V. album and V. laxum, respectively,whereas none of the samples of Arceuthobium andPhoradendron contained more than 3 mmol mol)1Chl.

The b-Car contents did not vary significantly fromthose of the species of Loranthaceae (Table 2), as wasthe case with Neo and VAZ, albeit the concentrationsfound in V. album and P. semivenosum were very low.Other than the difference in the extent of Lx accumu-lation described above, it seemed that there was notmuch difference between mistletoes in the two familieswith regard to the carotenoid composition.

Table 3 Carotenoid concentrations in mistletoe species of the family Viscaceae. The values are means of three to six samples ± SD(mmol/mol Chl a+b). For Phoradendron hexastichon, P. perrottetii, P. piauhyanum, P. semivenosum, and P. undulatum, only one samplewas analysed. Abbreviations are as defined in Table 2

Genus Species Neo V A Z VAZ DPS Lx Lut Lx/V b-Car S Caro

Viscum album 29±5 51±10 10±5 nda 61±12 0.2±0.1 2±3 58±12 0.0±0.1 34±8 184±27laxum 24±3 72±28 10±9 nd 81±23 0.1±0.1 6±3 106±50 0.1±0.1 66±14 282±67

Arceuthobium oxycedri 34±17 45±18 14±13 nd 59±5 0.3±0.2 1±1 121±40 0.0±0.0 157±80 379±91divaricatumb(female) 35±3 65±22 44±11 61±26 169±15 0.6±0.2 1±1 296±98 0.0±0.0 95±16 595±102

Phoradendron californicum 43±1 61±1 12±2.6 2±0 76±2 0.2±0.0 nd 240±3 – 76±1 435±5juniperinumb 52±9 55±10 14±10 nd 70±18 0.2±0.1 nd 334±54 – 90±24 545±103dipterum 50±7 97±3 3±3 nd 100±6 0.0±0.0 2±0 179±11 0.0±0.0 86±3 417±8emarginatum 57±1 66±6 4±2 nd 70±7 0.1±0.0 1±1 189±16 0.0±0.0 121±3 438±17piperoides 59±0 46±4 3±1 nd 49±5 0.1±0.0 2±1 225±18 0.1±0.0 85±6 419±9tunaeforme 55±3 59±10 4±4 nd 63±14 0.1±0.1 0±1 227±35 0.0±0.0 85±9 430±59hexastichon 53 84 3 nd 87 0.0 1 186 0.0 105 432perrottetii 47 68 4 nd 73 0.1 nd 176 – 83 380piauhyanum 48 85 7 nd 92 0.1 nd 210 – 72 423semivenosum 45 171 23 13 207 0.2 nd 290 – 19 560undulatum 38 75 75 187 337 0.8 nd 291 – 131 797

aConcentrations below the detection limit are shown as ndbSamples were not dark-adapted prior to the pigment analysis. For Arceuthobium divaricatum, only female plants were used for pigmentanalysis to avoid contamination with floral pigments (e.g. antheraxanthin), which would have been inevitable for male plants

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De-epoxidation of V and Lx

Out of the Loranthaceae and the two Viscum species, inwhich more than trace of Lx had been found, three plants(A. pendulum,Phth. ovata, andV. album)were subjected tohigh light treatment to examine the parallel operation ofthe two xanthophyll cycles (Fig. 2a–c). In all three speciesV and Lx underwent remarkably similar reduction uponlight exposure in much the same way as has been dem-onstrated in A. miquelii (Matsubara et al. 2001).

Comparison of the balance between the a- and b-Carbranches of the carotenoid biosynthesis pathway

The varying extents of Lx accumulation found in theLoranthaceae and the Viscaceae called for a closercomparison of the proportion of the pigment synthesisin the two branches of the carotenoid biosynthesispathway (Fig. 1). The question was whether the occur-rence of the Lx cycle is accompanied by increased pig-ment synthesis in the a-Car branch to yield greaterconcentrations of Lut, which may then become partlyavailable for the Lx cycle.

Figure 3 shows the profiles of carotenoid compositioninmistletoes of the two families based on the pigment datain Tables 2 and 3. The first three components from thebottom of each bar (Neo, VAZ, and b-Car) are synthe-sized in the b-Car branch whereas the upper two pigments(Lut and Lx) are in the a-Car branch. By comparing thetwo panels, it can be inferred that the operation of the Lxcycle would not be correlated with a large pool of a-Carbranch or Lut. The balance between the two branches diddiffer from plant to plant, but it was not related either tothe Lx concentrations or to the families.

Carotenoid composition in the Arabidopsis mutantlutOE

The relevancy of Lut availability to the induction of theLx cycle was further investigated by studying Lx

accumulation in the Arabidopsis transgenic plant lutOE(Fig. 4), in which synthesis of Lut is strongly enhancedas a result of an altered proportion of pigment

Fig. 3a, b Carotenoid compositions in mistletoe species of theLoranthaceae (a) and the Viscaceae (b; n=3–6). Neoxanthin (Neo)and the violaxanthin-cycle pigments (VAZ) are derived from b-carotene (b-Car) while lutein (Lut) and lutein epoxide (Lx) are froma-carotene. a-Carotene was detected only in Struthanthus margin-atus albeit in a small amount (not included in the figure)

Fig. 2a–c Light responses of violaxanthin (V) and lutein epoxide(Lx) in Amyema pendulum (a, n=5), Phthirusa ovata (b, n=3), andViscum album (c, n=3). Closed bars represent the originalconcentrations (=100%) obtained in a dark-adapted state. Openbars are concentrations after the high light treatment, expressed aspercent of the original level. High-light treatment was 1,700 lmolphotons m)2s)1 at 23 �C for 2 h for A. pendulum and 800 lmolphotons m)2 s)1 at 28 �C for 1.5 h for Phth. ovata and V. album

Fig. 4 Chromatogram of a leaf pigment extract of Arabidopsisthaliana lutOE (e-cyclase overexpressor). Fully expanded, matureleaves of plants grown under low light intensity (100 lmol photonsm)2 s)1) were used for pigment extraction. Values in parenthesesunder the names of carotenoids are the ratios of the averageconcentration in lutOE to wild type (n=20). The arrow marks atrace of Lx, whose level was not increased by altering the balance ofpigment biosynthesis in the a- and b-Car branch

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biosynthesis in the a- and b-Car branches by overex-pressing e-cyclase, a limiting enzyme of lutein synthesis(Pogson et al. 1996; Pogson and Rissler 2000; Fig. 1).Despite accumulation of more than 30% extra Lut, theLx peak recognized in the HPLC chromatograms wasminimal in 20 lines examined.

Since the synthesis of Lut in lutOE is increased at theexpense of V, we think it likely that some of the addi-tional Lut molecules are replacing V in the two V-binding sites of the light-harvesting antenna proteins,the peripheral V1 and internal L2 sites (Caffarri et al.2001). It has been proposed that either V in the V1 site(Ruban et al. 1999) or in both V1 and L2 sites (Caffarriet al. 2001) would be available for de-epoxidation byVDE. This means the binding of the additional Lut inthese sites did not initiate Lx accumulation in lutOE. Aquestion arises as to whether there are special bindingsites for the Lx-cycle pigments.

Pigment distribution within mistletoe thylakoids

In an effort to find the binding sites of Lx in mistletoethylakoids, in which the Lx cycle occurs, pigmentlocalization was investigated in different polypeptidefractions obtained by sucrose gradient ultracentrifuga-tion from solubilized thylakoid membranes of V. album.We used V. album for this experiment in spite of its smallLx-pool size (Table 3; Fig. 3b) because thylakoids of A.miquelii could not be fully solubilized with a milddetergent like a-DM. The use of a mild detergent wasnecessary to preserve pigments in their binding sites,especially those that were bound loosely. Mistletoethylakoids were fractionated into five bands and a pellet(Fig. 5). The pellet was loaded onto another sucrosegradient for purification before further analyses. Thepolypeptide composition of each fraction was identifiedby SDS–PAGE and absorption spectra as previouslydescribed (Dainese and Bassi 1991; Caffarri et al. 2001,data not shown).

Pigment distribution among different bands and thepolypeptide compositions are shown in Table 4. Since Aand Z were scarcely found in any of these bands obtainedfrom dark-adaptedV. album leaves (DPS=0.02), they arenot included in the table. A large portion of carotenoidpigments was found in the fraction of free pigments,particularly V and Lx, for which more than 50% wasfound inband 1.The relatively high concentrationsof freepigments (also Chl) compared with the observations insimilar studies (Verhoeven et al. 1999; Caffarri et al. 2001)could be due in part to the prolonged solubilization stepapplied forV. album samples (seeMaterials andmethods).

Nevertheless, our results are still consistent with thepresent-day view on the carotenoid localization inpigment–protein complexes of higher plant thylakoids(Yamamoto and Bassi 1996). It is clear that those pig-ments tightly bound in the protein interior (Neo, Lut, andChl) were less strongly represented in band 1 than V, themajority of which is in a peripheral site (V1 site) andtherefore can easily be removed during the sample prep-aration (Ruban et al. 1999; Verhoeven et al. 1999; Caffarriet al. 2001). Although all the xanthophyll pigments hadroughly the same distribution pattern, being mostly in thefraction of light-harvesting antenna complex (LHCII) ofphotosystem II (PSII), as opposed to the distribution ofb-Car which was concentrated in PSI and PSII, the closeresemblance between the distribution patterns of V andLx is readily apparent. Although the thylakoid solubili-zation was only partial for A. miquelii, data from theexperiment with A. miquelii also indicated Lx binding inbands 2 and 3 (S. Matsubara, T. Morosinotto, and R.Bassi, data not shown).

Carotenoid composition in other parasitic plants

Because the operation of the Lx cycle has also beenobserved in a holoparasitic dodder, Cuscuta reflexa(Bungard et al. 1999), a few more parasitic plants otherthan mistletoes were included in our pigment survey.Thesewere a dodder-like parasiteCassytha (Ca.)filiformisand two root parasites, E. cupressiformis and Me. pra-tense. As shown in Table 5,Ca. filiformis accumulated Lxto substantial levels (23±6 mmolmol)1Chl, or ca 20%ofV) while neither of the two root parasites exhibited a signof the Lx cycle. However, the lack of Lx in E. cupressi-formis provided an interesting clue to the phylogeny of theLx cycle inmistletoes; the genusExocarpos is amember ofthe family Santalaceae (sandalwood family) that belongsto the order Santalales, the same order as the Lorantha-ceae and the Viscaceae according to the currenttaxonomic classifications (Table 1).

Molecular phylogenetic analysis of the relationshipsof mistletoes in the Loranthaceae and the Viscaceae

In order to compare the Lx-cycle distribution withmistletoe phylogeny, we estimated the distances between

Fig. 5 Sucrose gradient profile of solubilized thylakoids fromdark-adapted leaves of Viscum album. Thylakoid samples werefractionated into five bands and a pellet. The pellet was furtherpurified by another sucrose gradient ultracentrifugation. Thepolypeptide composition of each fraction is given in Table 4together with pigment distribution

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different taxa of the two mistletoe families using18S rRNA genes as the molecular marker. We set E.bidwillii as the outgroup because of the relationship ofthe Santalaceae to both mistletoe families, albeit it isprobably closer to the Viscaceae than to the Lorantha-ceae (Kuijt 1969; Nickrent and Franchina 1990; Watson2001). For those species for which 18S rRNA gene se-quences were not available in the database, other specieswere chosen from the same genus so that all the generaincluded in the pigment study, except for Muellerina andNuytsia, could be represented.

A tree obtained by the neighbor joining method isshown in Fig. 6. The 11 mistletoes were divided into 2major groups in our 18S rRNA gene tree, 1 group with 4Loranthacean plants and the other with 7 Viscaceanplants. The latter was composed of three subgroups,namely Viscum, Arceuthobium, and Phoradendron, withViscum being the basal group. Most of the groups werestrongly supported by the bootstrap analysis (Fig. 6), asindicated near the base of each branch, except for themoderate 66% support for the branching of Arceutho-bium and Phoradendron. As a whole, the generic tree wasin good agreement with the systematic treatment of thesemistletoes. The bars next to the species’ names in Fig. 6indicate degrees of Lx accumulation. The distributionpattern of Lx accumulation matches the evolutionarylineages inferred from 18S rRNA gene sequences. Gen-erally, mistletoes in the Loranthaceae synthesized Lxwhile most of the plants in the Viscaceae did not. OnlyV. laxum and V. album, which belong to the basal groupof the Viscaceae branch (Fig. 6), exhibited slightlyhigher concentrations of Lx compared to other Visca-cean species (Table 3, Fig. 3).

Discussion

Epoxidase: a key enzyme for the occurrenceof the Lx cycle?

Juxtaposed with each other, species of the Loranthaceaeand Viscaceae displayed similar ranges of variation inrespect of Lut concentrations or the proportion of pig-ment synthesis in the a- and b-Car branch (Fig. 3),implying that the availability of Lut would not be alimiting factor for the occurrence of the Lx cycle. Sinceit has been shown that the synthesis of Lut can be lim-ited by e-cyclase activity (Pogson et al. 1996), weexamined the pigment composition in the Arabidopsise-cyclase overexpressor lutOE (Pogson and Rissler 2000)to determine if the Lx cycle can be induced by manip-ulation of pigment biosynthesis in the two branches ofthe carotenoid biosynthesis pathway. Overexpression ofthe e-cyclase in lutOE led to a >30% increase in Lutwith simultaneous reduction in V compared to wild type(Fig. 4). Despite this increased Lut, the amount of Lxdetected in lutOE was not affected, i.e. it remained attrace level. Because the extra Lut seemed to be synthe-sized at the expense of V in lutOE, it is likely that someof these additional Lut molecules are replacing V in theV-binding sites of the light-harvesting antenna proteins.This means that the additional Lut molecules bound inthese sites could be accessible for the xanthophyll cycleenzymes (Ruban et al. 1999; Caffarri et al. 2001);therefore, apoprotein steric hindrance for the interactionbetween ZE and Lut can be largely ruled out as a causeof the lack of Lx accumulation.

Table 4 Distribution of carotenoids and chlorophylls in different sucrose gradient bands obtained from dark-adapted leaves of Viscumalbum(DPS=0.02). Pigment amounts per fraction are given relative to the total of all fractions for each pigment (=100). Abbreviationsare as defined in Table 2

Band Content Neo V Lx Lut b-Car Chl a Chl b

1 Free pigments 20.7 52.9 55.4 33.8 22.5 9.0 3.12 CP29, CP26, CP24,

LHCII monomers11.1 11.3 7.2 9.2 7.1 13.6 11.5

3 LHCII trimers 60.1 24.3 22.3 47.4 5.0 44.3 71.94 CP29, CP24, LHCII

trimers, PSII8.1 5.3 3.1 6.0 13.0 11.7 9.6

5 PSII nda 0.9 1.4 0.5 10.7 4.7 0.4Pellet LHCI, PSI nd 5.4 10.6 3.1 41.7 16.7 3.5Total 100.0 100.0 100.0 100.0 100.0 100.0 100.0

aConcentrations below the detection limit are shown as nd

Table 5 Carotenoid concentrations in other parasitic plants examined in this study. The values are means of three samples ± SD (mmol/mol Chl a+b). Abbreviations are as defined in Table 2

Genus Species Neo V A Z VAZ DPS Lx Lut Lx/V b-Car S Caro

Cassytha filiformis 56±4 118±21 35±8 15±4 168±30 0.3±0.0 23±6 237±29 0.2±0.1 103±22 587±62Exocarpos cupressiformis 35± 1 118±2 8±1 nda 126±3 0.1±0.0 1±0 125±0 0.0±0.0 90±3 346±51Melampyrum pratense 25±2 40±2 7±1 nd 46±2 0.1±0.0 nd 65±2 – 86±4 221±10

aConcentrations below the detection limit are shown as nd

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The analysis of pigment localization in mistletoethylakoids (V. album) yielded no evidence for the pres-ence of special Lx-binding sites (Table 4). Instead, itsuggested that Lx could be sharing the same bindingsites with V. If Lx molecules are bound in the samebinding sites as V in plants with the Lx cycle, and if Lutin V-binding sites does not induce the Lx–Lut cycling inArabidopsis plants without the Lx cycle (Fig. 4), whatenables Lx synthesis in certain species? Our hypothesis isthat epoxidation from Lut to Lx is the limiting reactionfor Lx-cycle occurrence, and de-epoxidation of Lx backto Lut is possible once Lx is produced (Fig. 1).

This hypothesis is supported by our observation in anin vitro experiment that Lx was converted into Lut, inparallel with V into A and Z, by a recombinant Ara-bidopsis VDE expressed in E. coli (S. Matsubara,T. Morosinotto, and R. Bassi, unpublished data). Thiswas consistent with the previous reports on substratespecificity of lettuce and spinach VDE (Yamamoto andHigashi 1978; Grotz et al. 1999). In the study by Ya-mamoto and Higashi (1978), the lettuce VDE was foundto be able to convert Lx into Lut. For the spinach VDE,Lx was not tested but the enzyme was capable of de-epoxidizing capsanthin 5,6-epoxide, a monoepoxidewith a structure analogous to Lx apart from the secondring system (Grotz et al. 1999). Both Lx (= lutein 5,6-epoxide) and capsanthin 5,6-epoxide meet the proposedstructural requirements for VDE, that is, an all-transpolyene chain having at least one b-ionone ring with a3-hydroxy and 5,6 epoxy end group (Yamamotoand Higashi 1978). These results from in vitroexperiments, together with the observations of very

similar de-epoxidation rates of V and Lx in vivo (Fig. 2)as well as in situ (Matsubara et al. 2001), all suggest thatVDE could catalyse de-epoxidation reactions in the twoxanthophyll cycles in parallel. Furthermore, a recentstudy on red oak having the Lx cycle also demonstratedsimilar de-epoxidation rates for Lx and V (Garcıa-Plazaola et al. 2003).

In contrast to VDE with its specific function inphotoprotection, ZE has mostly been studied in relationto abscisic acid (ABA) biosynthesis (e.g. Marin et al.1996) because the same pathway of the b-Car branch isalso used for synthesis of this important plant hormonethat plays a role in seed development/dormancy and inplant stress response. The fact that only one copy of theZE gene has been found in Nicotiana plumbaginifolia(Marin et al. 1996), Chlamydomonas reinhardtii (Niyogiet al. 1997), Arabidopsis thaliana (Niyogi et al. 1998),and Lycopersicon esculentum (Thompson et al. 2000)indicates the dual roles of ZE in pigment/ABA synthesisand the operation of the V cycle. Substrate specificity ofZE has been examined by using enzyme cloned frompepper Capsicum annuum (Bouvier et al. 1996). Notably,it turned out that pigments with one b- and one e-ring,like Lut, are not epoxidized by the pepper ZE. Ittherefore seems that the structure of the second ring,which presumably is not critical for VDE, hinders thepepper ZE activity even though both VDE and ZE aremembers of the lipocalin family and share a conservedtertiary structure of a barrel configuration (Bugos et al.1998). The results from the study with the pepper ZEsuggest that the ZE enzyme would not catalyse theepoxidation of Lut to Lx, our putative limiting step ofthe Lx cycle.

How, then, does the Lx cycle occur in some species?This may be accounted for by mutation of ZE in theseplants. Such an assumption seems in agreement with ourcurrent understanding of molecular evolution of carot-enoid biosynthesis. Several carotenogenic enzymes havebeen classified as members of a gene family with differ-ent catalytic functions, presumably resulting from geneduplications (for review, see Hirschberg 2001). For in-stance, b- and e-cyclase, Neo synthase (Fig. 1), andcapsanthin–capsorubin synthase (an enzyme found inCapsicum) are believed to be derived from the b-cyclase.Mutation of ZE may have changed the substrate speci-ficity of the enzyme such that the structure of the secondring (= e-ring) does not impede pigment–enzymeinteraction, hence enabling synthesis of Lx from Lut. Itcan then be imagined that the newly synthesized Lxmolecules could have been bound to the light-harvestingantenna proteins (Table 4), which are known to beflexible in pigment binding (Kumagai et al. 1998; Green2001) as long as, for the case of carotenoids, a b-ringwith 3-hydroxy end group is provided (Phillip et al.2002).

Thus, parallel operation of the two xanthophyll cy-cles may have come into being, involving VDE, ZE and/or a homologous enzyme of ZE. Different epoxidationkinetics of the V and Lx cycles observed in a diurnal

Fig. 6 Phylogenetic tree based on 18S rRNA gene sequences ofsome mistletoe species and a root parasite Exocarpos bidwillii(Santalaceae) as the outgroup. The phylogeny was derived usingthe neighbor joining method. Since different distance measures(p distances, Jukes-Cantor, and Kimura 2-parameter) yielded thesame tree topology, the one obtained by Kimura 2-parameter isshown. The maximum likelihood method also resulted in identicaltopology. Bootstrap values are indicated near the base of eachbranch. The bars on the right-hand side of the species’ names showthe level of Lx accumulation in the genera within each clade: whiteno more than a trace amount of Lx, black appreciable amount ofLx, stripe small amount of Lx. Note that the species are differentfrom those in the pigment analysis albeit the same genera, exceptfor Muellerina and Nuytsia, which are not represented

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(Matsubara et al. 2001) as well as a seasonal time course(Matsubara et al. 2002) imply the involvement of dis-tinct epoxidase enzymes in the two cycles in mistletoes.Alternatively, the modified enzyme may catalyse theepoxidation reactions in both cycles albeit with differentaffinities to Z (and A) and Lut. Clearly, further researchon epoxidase from plants with the Lx cycle is needed.

Phylogeny of the Lx cycle in mistletoes

Our biochemical data on pigment composition of pho-tosynthetic tissues were in accord with the mistletoephylogeny estimated by 18S rRNA gene sequenceanalysis (Fig. 6). Taken together, these results stronglysupport the notion that the Loranthaceae and theViscaceae are distinct from each other, as has beenproposed previously based on classic criteria, such asfloral structures, embryology, and mutual affinities(Kuijt 1969), or biogeographical distributions (Barlow1983).

The pigment analysis revealed that Lx was almostabsent or accumulated only to small amounts in theViscacean species (Table 3, Fig. 3). In fact, the levels ofLx detected in Arceuthobium and Phoradendron werecomparable to those in other higher plants having onlythe V cycle (Young 1993b, or Fig. 4 forArabidopsis).Interestingly, Arceuthobium and Phoradendron havebeen regarded as evolutionarily advanced among mis-tletoe plants, judging from high mutation rates in thegenerally slowly evolving 18S rRNA (Nickrent andFranchina 1990; Nickrent et al. 1994) besides theirunique features of morphology, reproduction, anddispersal (Kuijt 1969). This implies that the ability tooperate the Lx cycle may have been lost (or deterio-rated) in many mistletoes of the Viscacean lineagesduring the evolution while it is conserved in theLoranthacean ones (Table 2, Fig. 3).

The Loranthaceae, on the other hand, has beenviewed as ancestral because of the occurrence of terres-trial root-parasitism (only in three genera, Nuytsia,Atkinsonia, and Gaiadendron) and epicortical roots (suchas in Muellerina), both of which are interpreted asremnants of terrestrial, non-parasitic lifestyles (Kuijt1969; Barlow 1983). Our analysis of 18S rRNA genesequences also indicated lower mutation rates amongmistletoes of the Loranthaceae than of the Viscaceae(Fig. 6), which is suggestive of slower evolution of theformer group. One can speculate that rapid mutations atthe molecular level in the species of the Viscaceae mayhave included the operational component(s) of the Lxcycle, resulting in the disappearance of the cycle, whilstit should be borne in mind that the phylogeny of thesmall-subunit rRNA gene, which represents the wholegenome of organisms, does not necessarily reflect thephylogeny of the genes for specific metabolic pathways(Xiong et al. 2000).

As an alternative possibility, it cannot be ruled outthat the Lx cycle may have appeared in the Lorantha-

ceae later in evolution. There is little doubt that somedegree of variability has been maintained throughout theevolution of the Loranthaceae, which is comprised of asmany as 950 species (Calder 1997). In a study on nuclearDNA variation in the Australian Loranthaceae, Martin(1983) pointed out the large increases in DNA content,mainly in the tropical and tropically derived genera, withconcomitant reduction in chromosome number, mean-ing an increase in the size of chromosomes. It has beenpostulated that this increase in chromosome size mayhave augmented recombination, and thus enhanced ge-netic diversity. Variability in DNA content was in factobserved in the Loranthacean species analysed in thisstudy. For instance, A. cambagei, A. miquelii, andA. pendulum (all x=9) contained more than twice asmuch DNA as M. eucalyptoides (x=11) of the genusMuellerina (Martin 1983), which presumably is a relic-tual genus (Barlow 1983). The DNA content of theprimitive root-parasitic mistletoe, N. floribunda (x=12),was only about 1/6 of that of A. miquelii (Martin 1983).However, the possibility of later appearance in theLoranthacean lineages seems rather unlikely if oneconsiders the high levels of Lx found in N. floribunda(Table 2) or the presence of the little but operative Lxcycle in V. album (Fig. 2c).

Given that the pool size of the Lx cycle undergoespronounced acclimation to the growth light environ-ment (Matsubara et al. 2001), it is difficult to directlyrelate the Lx concentrations measured in a small numberof samples to the evolutionary direction. Furtherinvestigations on the pigment composition in otherfamilies of Santalales could shed light upon the phy-logeny of the Lx cycle in mistletoes. Whichever theevolutionary direction could have been, based on thepattern of Lx distribution depicted in Fig. 6, we believethat genetic information of the key component(s) of theLx cycle would be inherited where it occurs. As dis-cussed above, ZE could be a possible candidate for it.

Why the Lx cycle?

Even with these new findings and the derived assump-tions, an important question about the physiologicalsignificance of the Lx cycle remains unanswered. Theoccurrence of the Lx cycle is neither ubiquitous among,nor restricted to, the mistletoe plants. Cuscuta reflexa(Bungard et al. 1999) and Ca. filiformis (Table 5), bothof which are chlorophyllous, non-mistletoe parasites,contain significant amounts of Lx. Likewise, the Lx cyclecannot be associated with a parasitic lifestyle in generalbecause Arceuthobium, Phoradendron, and the two rootparasites were devoid of Lx (Tables 3, 5). The originaldiscovery of the Lx cycle in green tomato fruit(Rabinowitch et al. 1975) as well as the finding indifferent oak species (Garcıa-Plazaola et al. 2002) alsoargues against an obligate relationship with parasitism.Moreover, another non-parasitic genus (Inga, familyFabaceae) has lately been added to the list of plants

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exhibiting the Lx cycle (S. Matsubara, C.J. Nichol,U. Rascher, and C.B. Osmond, unpublished data).

With the increasing number of species in the list of Lx-cycle plants, phylogenetic relationships, bywhich they canbe distinguished from the others lacking the Lx cycle,become less and less obvious. However, if ZE mutationwas the cause of the parallel operation of the two xan-thophyll cycles, it could have happened independently inseveral different groups of plants. In the carotenogenicenzymes, such mutation to one enzyme, resulting in cre-ation of a new enzyme with altered catalytic activity,seems to have occurred more than once (Hirschberg2001). Itmay have required nomore than a few changes tobring about modification in catalytic activity, as has beenevidenced by a recent report that activities of e-mono- andbi-cyclase can be switched by substitution of one singleamino acid (Cunningham and Gantt 2001). Close evolu-tionary relationships between some of the enzymes in thecarotenoid biosynthesis pathway can be inferred, forexample, from the fact that Neo synthase in tomato,whose amino acid sequence is 86.1% identical to cap-santhin–capsorubin synthase in pepper, shows activity ofb-cyclase (Ronen et al. 2000), from which both enzymesare supposed to have evolved (Bouvier et al. 2000).

Thus far, we have not come up with any ecophysio-logical or evolutionary factors that are common to theLx-cycle plants and could possibly explain the significance ofoperating this additional cycle in parallel with theV cycle.Although a photoprotective role of theLx cycle for leavesor plants growing in the shade has been postulated(Garcıa-Plazaola et al. 2002, 2003), there is some dis-crepancy between the distribution of the Lx cycle inhigher plant species and the light environments in theirnatural habitats. The raison d’etre of the Lx cycle stillremains open to speculations and awaits future investi-gations.

Acknowledgements S.M. is the recipient of an ANU PhD Grad-uate School Scholarship (Endowment for Excellence) and anOverseas Postgraduate Research Scholarship. Part of this studywas supported by a Forschungspreis from the Alexander vonHumboldt Stiftung to C.B.O. and PRONEX-CNPq Brazil toA.C.F.. We thank Dr. Georg Weiller (Canberra, Australia) forinvaluable advice on the phylogenetic analyses and Dr. Marilyn C.Ball for helpful comments on the manuscript. Kind assistance forHPLC experiments by Prof. Carlos A. Schwartz and Dr. OsmindoR. Pires Jr. (Brasilıa, Brazil) and the identification of species in Riode Janeiro by Carlos H. R. de Paula (Rio de Janeiro, Brazil) aregreatly acknowledged. Plant materials were collected with the helpof Claudenir S. Caires and Vandelio C. Mendes (Brasılia, Brazil),Dr. Cornelia Buchen-Osmond (Oracle, USA), Dr. Stephan Sch-neckenburger (Darmstadt, Germany), and Dr. John C.G. Banksand Dr. Torsten Julich (Canberra, Australia), to whom we wouldlike to extend the warmest thanks.

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