Photoprotection of reaction centres in photosynthetic organisms: mechanisms of thermal energy dissipation in desiccated thalli of the lichen Lobaria pulmonaria Ulrich Heber 1 , Wolfgang Bilger 2 , Roman Tu ¨rk 3 and Otto L. Lange 1 1 Julius-von-Sachs-Institute of Biological Sciences, University of Wu ¨rzburg, D-97082 Wu ¨rzburg, Germany; 2 Botanical Institute, University of Kiel, D-24098 Kiel, Germany; 3 Department of Organismic Biology, University of Salzburg, A-5020 Salzburg, Austria Author for correspondence: Wolfgang Bilger Tel: +49 431 880 4289 Email: [email protected]Received: 17 August 2009 Accepted: 8 September 2009 New Phytologist (2009) doi: 10.1111/j.1469-8137.2009.03064.x Key words: chlorophyll fluorescence, conformational changes, energy dissipation, lichens, photoprotection, photosystem II, reaction centre. Summary • The photobionts of lichens have previously been shown to reversibly inactivate their photosystem II (PSII) upon desiccation, presumably as a photoprotective mechanism. The mechanism and the consequences of this process have been investigated in the green algal lichen Lobaria pulmonaria. • Lichen thalli were collected from a shaded and a sun-exposed site. The activa- tion of PSII was followed by chlorophyll fluorescence measurements. • Inactivation of PSII, as indicated by the total loss of variable fluorescence, was accompanied by a strong decrease of basal fluorescence (F 0 ). Sun-grown thalli, as well as thalli exposed to low irradiance during drying, showed a larger reduction of F 0 than shade-grown thalli or thalli desiccated in the dark. Desiccation increased phototolerance, which was positively correlated to enhanced quenching of F 0 . Quenching of F 0 could be reversed by heating, and could be inhibited by glutaral- dehyde but not by the uncoupler nigericin. • Activation of energy dissipation, apparent as F 0 quenching, is proposed to be based on an alteration in the conformation of a pigment protein complex. This per- mits thermal energy dissipation and gives considerable flexibility to photoprotec- tion. Zeaxanthin formation apparently did not contribute to the enhancement of photoprotection by desiccation in the light. Light-induced absorbance changes indicated the involvement of chlorophyll and carotenoid cation radicals. Abbreviations: F 0 , minimum fluorescence, Q A in the RC of PSII oxidized; F m , max- imum fluorescence, Q A reduced; F s , stationary fluorescence, Q A partially reduced; F v , variable fluorescence, F m ¢-F s or F m -F 0 ; NPQ, nonphotochemical fluorescence quenching, F m ⁄ F m ¢ – 1 or, in desiccated thalli, F m ⁄ F 0 desiccated ) 1 because F m is very close to F 0 desiccated ; PPFD, photosynthetic photon flux density; PSI, photosys- tem I; PSII, photosystem II; Q A, quinone acceptor in the reaction centre of PSII; RC, reaction centre. Introduction Many lichens are desiccation-resistant as they survive peri- ods of total dehydration (see Nash, 2008). Moreover, in contrast to hydrated and metabolically active lichens, most dry lichens tolerate long-lasting high incident light intensi- ties without suffering from photooxidative damage and pigment destruction (Demmig-Adams et al., 1990). With regard to their ability to survive desiccation and prevent photooxidative damage in this state, they differ from the majority of higher plants. In natural open habitats, lichens normally dry out as soon as supply of moisture ceases. In high mountain regions, dry saxicolous lichens are exposed for long periods of time to strong light. Desert lichens may be hydrated for < 10% of their total life span. During most of their life they persist in a dry state of dormancy under New Phytologist Research ȑ The Authors (2009) Journal compilation ȑ New Phytologist (2009) New Phytologist (2009) 1 www.newphytologist.org
Transcript
Photoprotection of reaction centres in photosyntheticorganisms: mechanisms of thermal energy dissipationin desiccated thalli of the lichen Lobaria pulmonaria
Ulrich Heber1, Wolfgang Bilger2, Roman Turk3 and Otto L. Lange1
1Julius-von-Sachs-Institute of Biological Sciences, University of Wurzburg, D-97082 Wurzburg, Germany; 2Botanical Institute, University of Kiel,
D-24098 Kiel, Germany; 3Department of Organismic Biology, University of Salzburg, A-5020 Salzburg, Austria
Fv, variable fluorescence, Fm¢-Fs or Fm-F0; NPQ, nonphotochemical fluorescence
quenching, Fm ⁄ Fm¢ – 1 or, in desiccated thalli, Fm ⁄ F0 desiccated ) 1 because Fm is
very close to F0 desiccated; PPFD, photosynthetic photon flux density; PSI, photosys-
tem I; PSII, photosystem II; QA, quinone acceptor in the reaction centre of PSII;
RC, reaction centre.
Introduction
Many lichens are desiccation-resistant as they survive peri-ods of total dehydration (see Nash, 2008). Moreover, incontrast to hydrated and metabolically active lichens, mostdry lichens tolerate long-lasting high incident light intensi-ties without suffering from photooxidative damage andpigment destruction (Demmig-Adams et al., 1990). With
regard to their ability to survive desiccation and preventphotooxidative damage in this state, they differ from themajority of higher plants. In natural open habitats, lichensnormally dry out as soon as supply of moisture ceases. Inhigh mountain regions, dry saxicolous lichens are exposedfor long periods of time to strong light. Desert lichens maybe hydrated for < 10% of their total life span. During mostof their life they persist in a dry state of dormancy under
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daily high light intensities. Recent experiments have shownthat lichens are even able to survive dryness, heat or cold,and incident radiation for 14 d when openly exposed to theextraterrestrial space (Sancho et al., 2007). Thus, resistanceto light of such species seems to be rather unlimited in prin-ciple. However, Gauslaa & Solhaug (1999, 2001) also dem-onstrated that light can damage lichens while they are dry.This leads to the conclusion that high photoresistance ofdry lichens is not a general feature of thallus dehydration.Rather, specific mechanisms must be involved to facilitateprotection against excessive light. In the present contribu-tion, which is a continuation of previous work (Kopeckyet al., 2005; Heber et al., 2006a,b, 2007; Heber, 2008), weinvestigate such mechanisms. As before, we use fluorescenceresponses to desiccation and hydration in order to obtaininformation on energy dissipation. We compare thalli ofthe foliose lichen Lobaria pulmonaria that have grown inthe deep shade with those that are adapted to full sunlight,because it is known that differences in growth conditionsresult in differences in phototolerance. L. pulmonaria isespecially suited for our experiments because much infor-mation about its performance has been obtained by Gauslaaand his group and is available in the literature (Gauslaa &Solhaug, 1999, 2000, 2001; McEvoy et al., 2007; Stepigovaet al., 2008). L. pulmonaria uses a xanthophyll cycle to syn-thesize zeaxanthin in the light. Zeaxanthin plays a centralrole in the photoprotection of many plants (Demmig-
Adams, 1990). In our work, we have tried to answer the fol-lowing questions. Can we explain differences in phototoler-ance between thalli from sun-exposed and thalli from deeplyshaded habitats? What are the mechanisms that contributeto photoprotection? How are they activated? How importantis the well-investigated mechanism of zeaxanthin-dependentenergy dissipation for the photoprotection of L. pulmonaria?
Materials and Methods
Sun-exposed thalli of the lichen Lobaria pulmonaria (L.)Hoffm. were obtained from the bark of an isolated tree ofAcer pseudoplatanus at Ramsau, Germany, not far from Ber-chtesgaden, at an altitude of 800 m. This tree is exposed tothe sun during all seasons; the lichen specimens were col-lected from the side exposed to the south ⁄ south-west. Thalligrown in the deep shade were collected from the bark ofwillow shrubs (Salix sp.) to the south of the Hintersee lakeat an altitude of 800 m. The specimens grew 60 cm abovethe soil in the deep shade of the closed canopy of Salix sp.and Picea abies. The thalli from the sun-exposed site weredistinctly brownish, whereas the shade-grown samples weregreyish-green. Dates of collection were 27 July 2005 and 26January 2008. The samples were sent to Wurzburg in thedesiccated state, where they were stored at )20�C. No dete-rioration of fluorescence signals was observed over the stor-age period. Experiments in Figs 1, 4 and 7 were done in
a1 a2 a3
b1 b2 b3
Fig. 1 Modulated chlorophyll fluorescence during three cycles of hydration and slow desiccation of thalli of sun-grown (a1, a2, a3) and shade-grown (b1, b2, b3) Lobaria pulmonaria. Hydration (addition of H2O) increased fluorescence reversibly. Very strong or, alternatively, very weaklight pulses were given every 500 s to probe for charge separation in photosystem II (PSII) reaction centres (RCs). The few strong light pulses(photosynthetic photon flux density (PPFD) = 11 000 lmol m)2 s)1) given during the first and third hydration cycle are recognizable by largetransient fluorescence responses. They were intended to indicate the maximal variable fluorescence Fv = (Fm ) F0). Actinic light was absentand light pulses were weak (PPFD = 2 lmol m)2 s)1, recognizable by small fluorescence spikes) during most of the first and third hydrationcycles. Actinic light was present (PPFD = 300 lmol m)2 s)1) during most of the second hydration cycle where all light pulses given werestrong. Sensitivity of fluorescence recording was identical in (a) and (b) so that fluorescence intensities can be compared. m.b., measuringbeam. For further explanation see text.
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March and November 2008 with the samples from 2008;the remaining experiments were done during November2007 with the samples from 2005.
Prolonged dark adaptation (36 or 48 h) of hydrated thalliwas intended to minimize zeaxanthin concentrations. Zea-xanthin is known to be converted to violaxanthin in thedark or in low light (Hager, 1980; Bjorkman & Demmig-Adams, 1994). The thalli were slowly dried in the dark atroom temperature in air of a relative humidity below 60%(equivalent to a water potential below )70 MPa). Usually,a very low intensity modulated red measuring beam with anaverage photosynthetically active photon flux density(PPFD) of 0.04 lmol m)2 s)1 was present during slowdrying to enable the recording of fluorescence emission.Absence of activity of zeaxanthin-dependent energy dissipa-tion in predarkened hydrated thalli was checked by makingsure that quenching of basal or F0 chlorophyll fluorescencewas not induced by illumination with strong light pulseswhich lasted 1 s (Katona et al., 1992; Kopecky et al., 2005;Heber et al., 2006b). Active zeaxanthin-dependent energydissipation is indicated by a short and transient postillumi-nation F0 quenching response which was carefully distin-guished from postillumination oxidation of reduced QA
(quinone acceptor) in reaction centres (RCs) of photosys-tem II (PSII).
Modulated chlorophyll fluorescence was measuredbeyond 700 nm after excitation at c. 650 nm (using the far-red transmitting filter RG 9; Schott, Mainz, Germany) by apulse amplitude modulation fluorometer (PAM 101; Walz,Effeltrich, Germany) (Schreiber et al., 1986).
Short pulses (usually 1 s) of white light (Calflex c andDT-Cyan filters; Balzers, Liechtenstein) from a halogenlamp (KL 1500; Schott) were brought to the cuvette by fibre-optics to probe for variable fluorescence. The PPFD of thelight pulses was usually c. 10 000 lmol m)2 s)1 (but only 2or 4 lmol m)2 s)1 when high light fluxes had to beavoided). Actinic light was provided by a second halogenlamp. Heat- and far-red-absorbing filters were a com-bination of Calflex c and DT-Cyan. For light stress experi-ments with desiccated thalli lasting > 30 min, strong lightfrom either a halogen or a mercury lamp was used(PPFD £ 1500 lmol m)2 s)1). At comparable PPFDs (asmeasured by a Li-Cor 189 quantum sensor; Walz), themercury lamp (Osram Powerstar HQI-R, 250 W; OsramGmbH, Munchen, Germany) produced more photodamagethan the halogen lamp. Whenever necessary, the temperatureof the samples was monitored by a thermocouple. Heatingexperiments and experiments with gas mixtures of differentCO2 content were performed using a sandwich-type cuvettewhich permitted controlled gas flow over the thalli.
Light-dependent absorption changes at 800 nm weremeasured in reflection using the PAM fluorometer in com-bination with an ED800 T emitter ⁄ detector unit (Walz).This attachment was modified for the measurement of
absorption changes at 950 nm by replacing the originalLED of the emitter ⁄ detector unit with an LED with peakemission at 950 nm.
For carotenoid determinations, desiccated L. pulmonariathalli were stored in a freezer at )20�C. Before extraction,they were washed twice with 100% acetone, subsequentlybriefly rewetted in 20 mM Hepes solution in darkness andthen ground in liquid nitrogen in a mortar before adding80% acetone ⁄ 20% 20 mM Hepes. After centrifugation,pellets were extracted twice with 100% acetone. Combinedextracts were analysed for carotenoids and chlorophyll aand b using an Agilent 1100 HPLC system with diode arraydetection at 445 and 407 nm following essentially themethod described in Niinemets et al. (1998).
Glutaraldehyde was obtained from Sigma-Aldrich ⁄ Fluka(Seelze, Germany).
Results
Changes in emission of chlorophyll fluorescence dur-ing desiccation and hydration
Lichens respond to desiccation and hydration by largechanges in fluorescence emission (Lange et al., 1989).Figure 1 shows such changes for thalli of L. pulmonaria inboth hydrated and desiccated states. The thalli had beencollected in late January under alpine winter conditions. Onaddition of water to desiccated thalli, chlorophyll fluores-cence increased more in sun-exposed than in shade-grownthalli. Slow drying after hydration resulted in greater loss offluorescence in sun-exposed than in shade-grown thalli.When not caused by altered light absorption, loss of fluores-cence indicates activation of energy dissipation because fluo-rescence and thermal energy dissipation compete directlyfor the energy of absorbed light when photochemical use oflight energy is negligible, as is the case in the absence ofwater. Greater desiccation-induced fluorescence quenchingis equivalent to greater thermal dissipation of absorbed lightenergy. Greater loss of fluorescence upon desiccation ofsun-grown vs shade-grown L. pulmonaria therefore suggestsbetter photoprotection of sun-grown than of shade-grownL. pulmonaria.
The extent of the increase of fluorescence observed onhydration depended on the previous history of the thalli. In
Table 1 Ratios F0 hydrated ⁄ F0 desiccated in the experiments of Fig. 1
Sun-exposed Shade-grown
F0 ⁄ F0¢¢, dark-dried,after first hydration
4.8 2.5
F0 ⁄ F0¢¢¢, light-dried,after second hydration
12.9 3.7
F0 ⁄ F0¢¢¢¢, dark-dried,after third hydration
4.8 2.7
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the experiment illustrated in Fig. 1(a), basal fluorescence,F0¢, of sun-grown desiccated L. pulmonaria increased onhydration to the value termed F0. The ratio F0 ⁄ F0¢ was 7.7.After slow drying in near darkness (the very weak measuringbeam needed for the measurement of fluorescence had aPPFD of only 0.04 lmol m)2 s)1), the ratio F0 ⁄ F0¢decreased to 4.8 (Table 1). Nonphotochemical fluorescencequenching (NPQ) had been 9 before drying and was 5.5afterwards. However, after the same thallus was hydrated asecond time, and then dried slowly in light ofPPFD = 300 lmol m)2 s)1, which is c. 20% of full sun-light, F0¢ was lowered to F0¢¢¢. The ratio F0 ⁄ F0¢¢¢ was now12.9, and NPQ was 15. A third hydration returned fluores-cence almost to the initial F0 intensity. Slow drying underthe same conditions of near darkness as during the first dry-ing returned the ratio F0 ⁄ F0¢¢¢¢ to the previous ‘dark’ valueof 4.8. In other words, drying the thallus in near darknessdid not suppress F0 as much as it did during drying in thepresence of illumination. Not much illumination duringslow drying was needed to observe this effect. It was similarwith PPFDs of 300, 130 and 25 lmol m)2 s)1 (data notshown). Increased fluorescence quenching after desiccationof L. pulmonaria in the light has recently also been observedby Stepigova et al. (2008).
Strong 1 s light pulses of PPFD = 11 000 lmol m)2 s)1
(equivalent to more than six times full sunlight) failed tochange fluorescence appreciably when the thalli were desic-cated. Similar observations were made before with differentalgal and cyanobacterial lichens (Lange et al., 1989). After afew minutes’ hydration, strong pulses increased fluorescenceto the Fm value. Fv ⁄ Fm was 0.26 shortly after the first hydra-tion, that is, less than the maximum quantum efficiencycommonly observed in predarkened leaves, where Fv ⁄ Fm
ratios are c. 0.8 (Bjorkman & Demmig, 1987), and alsolower than the values 0.63–0.76 that are usually found afterprolonged dark adaptation in lichen photobionts (Jensen,2002). Transient postillumination F0 quenching immedi-ately after strong light pulses indicated the induction ofzeaxanthin-dependent energy dissipation by the light pulses(Katona et al., 1992; Heber et al., 2007). This effect disap-peared during the experiment as shown by the absence ofpostillumination F0 quenching when a strong light pulsewas given later on. Charge separation in PSII RCs was stillsignificant after strong light pulses were replaced by veryweak light pulses of PPFD = 2 lmol m)2 s)1. Under theweak pulses, fluorescence responses were much reduced. Asdrying progressed, charge separation decreased until it wasno longer noticeable.
The second hydration increased fluorescence and re-established charge separation but the strong light pulsesfailed to be followed by postillumination F0 quenching thistime, showing that zeaxanthin-dependent energy dissipationhad been slowly inactivated under the near-darkness condi-tions of the first hydration. When continuous illumination
with PPFD = 300 lmol m)2 s)1 was added together withthe strong light pulses, the transient fluorescence peaks elic-ited by the light pulses decreased to Fm¢. The lowering ofFm to Fm¢ is attributed to nonphotochemical fluorescencequenching. The purpose of illumination had been to acti-vate zeaxanthin-dependent energy dissipation. In the pres-ence of actinic illumination, some RCs were closed and didnot contribute to the fast pulse-induced fluorescenceresponses. As water continued to be lost, not only stationaryfluorescence but also pulse-induced fluorescence responsesdecreased. The final fluorescence intensity after drying hadbeen complete was below that observed after the first hydra-tion. Apparently, illumination during hydration had in-creased fluorescence quenching. This confirms observa-tions reported in a recent publication of Stepigova et al.(2008).
In principle, the third hydration returned the thallus tothe situation observed during the first hydration except forone important point. Immediately after the first lightpulses, small transient postillumination quenching effectswere observed which were absent after the first hydration.Apparently, zeaxanthin-dependent energy dissipation hadbeen activated during the second hydration. Postillumina-tion F0 quenching slowly disappeared during hydration asshown by its absence when a strong light pulse were givenlater on during the hydration phase. It seems that zeaxan-thin-dependent energy dissipation was slowly lost under thenear-darkness condition of the third hydration. The samehad been observed during the first hydration.
Figure 1(b) shows, for desiccated shade-grown L. pulmo-naria, the same procedure described for sun-grown L. pul-monaria in Fig. 1(a). The sensitivity of measurements wasidentical in Figs 1(a) and (b). Three differences need to beemphasized, as follows. First, F0¢ fluorescence intensitieswere higher in shade-grown than in sun-adapted L. pulmo-naria, but ratios F0 ⁄ F0¢ were lower (see Table 1); NPQ val-ues were lower than in sun-grown L. pulmonaria, suggestingthere was less desiccation-induced energy dissipation. Sec-ondly, continuous illumination during the second hydra-tion decreased pulse-induced fluorescence responses more,but stationary fluorescence less, than in sun-grown L. pul-monaria. And thirdly, during the third hydration, quantumefficiencies of charge separation as expressed by Fv ⁄ Fm val-ues were 12% below the values measured during the firsthydration. This decline suggested damage to the RCs whilethe shade-grown L. pulmonaria had received a PPFD of300 lmol m)2 s)1 during the second hydration phase.
Table 1 compares the ratios F0 hydrated ⁄ F0 desiccated obs-erved in the experiment whose results are shown in Fig. 1.Desiccation-induced quenching of fluorescence was stron-ger in sun-grown than in shade-grown L. pulmonaria,whether or not the thalli were dried in darkness or in thelight. The presence of light during desiccation increased theF0 hydrated ⁄ F0 desiccated ratios.
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Inhibition of desiccation-induced loss of fluorescenceby glutaraldehyde
The amplitude of chlorophyll fluorescence is a product oflight absorption by photosynthetic pigments and the quan-tum yield of fluorescence emission. The latter is decreasedby the activation of a competitive reaction such as nonradia-tive energy dissipation. Desiccation of various lichens, andin particular of L. pulmonaria (Gauslaa & Solhaug, 2001),has been reported to decrease light transmission to the algallayer (Dietz et al., 2000; Heber et al., 2007). There was aquestion as to what extent this affects the desiccation-induced fluorescence quenching shown in Fig. 1. Glutaral-dehyde has been shown to inhibit desiccation-induced fluo-rescence quenching in chlorolichens such as Parmeliasulcata and Cladonia rangiformis (Heber et al., 2007; Heber,2008). It possesses two reactive aldehyde groups capable ofreacting with proteins but does not alter light absorption bypigments. Figure 2 shows emission of modulated chloro-phyll fluorescence from sun- (Fig. 2a) and shade-grown
(Fig. 2b) L. pulmonaria after 1 h preincubation in 0.25%glutaraldehyde while the thalli were slowly dehydrated.Background light had a PPFD of 4 lmol m)2 s)1. Stronglight pulses given every 500 s increased fluorescence tran-siently, thereby revealing residual light-dependent chargeseparation. During progressive loss of water, fluorescencedecreased and pulse-induced fluorescence responses chan-ged direction as desiccation neared completion. Fluores-cence emission from desiccated glutaraldehyde-treated thalliwas stronger than fluorescence emission from desiccatedthalli in the experiments of Fig. 1. The ratio F0 hydrated ⁄F0 desiccated was 1.34 in Fig. 2(a) and 1.32 in Fig. 2(b). Thiscompares with the much larger F0 hydrated ⁄ F0 desiccated ratioslisted in Table 1. The loss of fluorescence still observedin Fig. 2 after desiccation is presumably attributable eitherto incomplete inhibition of fluorescence quenching byglutaraldehyde or to loss of light absorption during desicca-tion, or a combination of both.
Heat-induced increase of fluorescence emission indesiccated thalli
Recently, it was suggested that desiccation of lichens chan-ged the conformation of a pigment protein complex so as tocreate dissipating centres within the complex (Heber et al.,2007; Heber, 2008). Heating inactivates proteins byunfolding secondary and tertiary protein structures. Whenthe desiccated lichen P. sulcata was heated, chlorophyll fluo-rescence increased and charge separation in PSII RCs wasre-established as indicated by the appearance of pulse-induced fluorescence spikes (Heber & Shuvalov, 2005).
(a)
(b)
Fig. 2 Modulated chlorophyll fluorescence of sun- (a) and shade-grown (b) Lobaria pulmonaria as affected by slow desiccation.Before the experiment, the thalli were incubated for 1 h with 0.25%glutaraldehyde. Fluorescence was elicited by an average photosyn-thetic photon flux density (PPFD) of 4 lmol m)2 s)1. Very stronglight pulses were given every 500 s to probe for charge separation inphotosystem II (PSII) reaction centres (RCs). Note the reversal ofdirection of pulse-induced fluorescence responses during drying.m.b., measuring beam.
(a) (b)
(c) (d)
Fig. 3 Changes of fluorescence of desiccated sun-grown (a, b) andshade-grown (c, d) Lobaria pulmonaria during heating and cooling.Heating rate was c. 2�C min)1 and the actual temperature (�C) isindicated on top of the fluorescence trace. Hydrated thalli were pre-darkened for 36 h. They were then dried slowly either in the dark (a,c) or in light with a photosynthetic photon flux density (PPFD)of 25 lmol m)2 s)1 (b, d). Measurements were done in darkness,and strong light pulses were given every 200 s.
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Figure 3 shows that increasing the temperature of desic-cated L. pulmonaria thalli decreased fluorescence slightlybetween 10 and 30�C before it increased strongly above60�C until no further increase was observed close to 80�C.Importantly, the heat-induced fluorescence increase was lar-ger in thalli that had been dried in low light (Fig. 3b,d)than in thalli dried in the dark (Fig. 3a,c). The heat-induced increase was particularly large in a sun-grown andlight-dried thallus (Fig. 3b), which had lost more fluores-cence on desiccation than a thallus dried in the dark(Fig. 1a). The data suggest that the desiccation-inducedquenching of chlorophyll fluorescence can be reversed byheating desiccated thalli. However, in contrast to previousobservations with P. sulcata (Heber & Shuvalov, 2005), var-iable fluorescence could not be restored upon heating of L.pulmonaria. Only some photochemical activity appeared athigh temperature, as indicated by the reversible fluorescencequenching induced by strong light pulses (Fig. 3). The dif-ference between P. sulcata and L. pulmonaria was probablythe result of the higher heat sensitivity of the latter (Gauslaa& Solhaug, 1999).
Zeaxanthin cycle and zeaxanthin-dependent energydissipation in L. pulmonaria
Increased desiccation-induced quenching under illumina-tion suggested a role of zeaxanthin in energy dissipationbecause zeaxanthin is known to be synthesized in the lightfrom violaxanthin (Demmig-Adams, 1990). When present,it contributes to photoprotection under the control of thePsbS protein of the thylakoids (Li et al., 2004; Takizawaet al., 2007). Components of the xanthophyll cycle (zeaxan-thin, violaxanthin and antheraxanthin) were measured indesiccated thalli that had been predarkened in the hydratedstate for 36 h and were then slowly desiccated either indarkness or in light of PPFD = 300 lmol m)2 s)1. Resultsare shown in Table 2. They reveal that drying of hydratedsun-grown L. pulmonaria in the light increased concentra-tions of zeaxanthin at the expense of violaxanthin. Zeaxan-thin decreased after drying in the dark, whereas viola-xanthin increased. Very similar observations were made
with shade-grown L. pulmonaria, but zeaxanthin increasedless during drying in the light than in sun-grown L. pulmo-naria. Even predarkening of hydrated L. pulmonaria for36 h did not eliminate zeaxanthin fully.
The data show functioning of the xanthophyll cycle butdo not prove that activation of zeaxanthin-dependentenergy dissipation by light during hydration is necessary forphotoprotection of subsequently desiccated L. pulmonaria.Activation needs protonation of the PsbS protein (Li et al.,2004; Takizawa et al., 2007). Although, normally, light-dependent proton transport into thylakoids is responsiblefor activating zeaxanthin-dependent energy dissipation,20% CO2 in air, acting as a potential acid, has also beenshown to be effective in activating energy dissipation inlichens and mosses when zeaxanthin is present but light isabsent (Bukhov et al., 2001). No quenching was observedin the absence of zeaxanthin. Figure 4 shows that even after48 h predarkening, considerable quenching of fluorescencewas produced by 20% CO2 in hydrated L. pulmonaria.Quenching was stronger in sun-grown predarkened thallithan in shade-grown thalli. The ratio of stationary fluores-cence Fs to quenched fluorescence F¢ was 1.8 in shade-grown L. pulmonaria and 4.5 in sun-grown L. pulmonaria.Brief transient postillumination loss of fluorescence wascaused by postillumination oxidation of reduced QA, not byF0 quenching. The experiments not only confirmed thepresence of zeaxanthin in the thalli even after prolongedpredarkening but also the necessity of protonation for theactivation of zeaxanthin-dependent energy dissipation.Moreover, they showed persistence of light-dependentcharge separation in the presence of energy dissipation acti-vated by CO2. The ratio (Fm ) F) ⁄ Fm, which serves to indi-cate charge separation in PSII RCs, decreased under theinfluence of 20% CO2 by 25% in shade-grown L. pulmona-ria and by 35% in sun-grown L. pulmonaria. CO2-inducedNPQ was 1.8 in shade-grown and 6.8 in sun-grown thalli.CO2 did not alter fluorescence emission in desiccated L.pulmonaria (data not shown).
To answer the question as to whether activation of zea-xanthin-dependent energy dissipation is responsible for thelight effect on fluorescence quenching during desiccation, as
Table 2 Antheraxanthin (A), zeaxanthin (Z),violaxanthin (V) and the sum of antheraxan-thin + zeaxanthin + violaxanthin (VAZ) indesiccated Lobaria pulmonaria (thalli col-lected in summer)
Z A V VAZ
L. pulmonaria, grown in the sunDried in the light1 113 ± 7 18 .1 ± 3.2 18.1 ± 0.7 149 ± 3.5Dried in darkness 21.4 ± 3.5 15.2 ± 4.7 87.2 ± 11.3 124 ± 11.8Light ⁄ dark �5 �1.2 �0.2
L. pulmonaria, grown in the shadeDried in the light1 47 ± 11.9 13.6 ± 4.7 22.6 ± 2.2 83.3 ± 14.2Dried in darkness 28.3 ± 15.6 11.8 ± 2.9 54.3 ± 4.5 94.3 ± 17.4Light ⁄ dark �2 �1.2 �0.4
Contents in mmol mol)1 chlorophyll; means of five determinations ± SD.1Photosynthetic photon flux density (PPFD) = 300 lmol m)2 s)1.
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shown in Fig. 1, attempts were made in the experiment ofFig. 5 to interfere with the control of zeaxanthin-dependentenergy dissipation by the PsbS protein. Protonation of thePsbS protein requires a lowering of the intrathylakoid pHby proton-coupled light-dependent electron transport. Thiscan be prevented by the protonophore nigericin, which isalso an effective inhibitor of zeaxanthin synthesis in thelight. In the experiment of Fig. 5, part of a sun-grownhydrated thallus was predarkened for 36 h to decrease zea-xanthin concentrations (see Table 2). It was then slowlydried in the dark and subsequently hydrated in 5 lM nige-ricin. Effectiveness of the inhibition of intrathylakoid
proton deposition by nigericin was demonstrated by thesensitive response of chlorophyll fluorescence to low light(PPFD = 2 lmol m)2 s)1) which raised fluorescence tran-siently to the Fm value in the presence of nigericin, but notin its absence (see Fig. 1). This indicates inhibition of elec-tron transport to a large extent, probably because of block-age of photosynthesis as a result of inhibition of photo-phosphorylation. Slow drying of the nigericin-treatedhydrated thallus under illumination with PPFD =300 lmol m)2 s)1 quenched fluorescence as much as it didin an untreated illuminated control thallus and more thanin a predarkened thallus which had been dried in near dark-ness. The ratio F0 ⁄ F0¢ was 9.1 in the experiment of Fig. 5and the ratio Fm ⁄ F0¢¢ 14.8. The corresponding ratios in acontrol experiment without nicericin were 8.7 and 16.3. Anigericin experiment similar to that shown in Fig. 5 wasalso performed with the chlorolichen C. rangiformis (Heber,2008). The results were similar to those of the L. pulmona-ria experiment of Fig. 5.
Irreversible loss of fluorescence and of light-depen-dent charge separation in desiccated L. pulmonariaunder prolonged strong illumination
Figure 6 shows representative examples of light stress experi-ments. They are intended to give information on the sensi-tivity of desiccated L. pulmonaria to sunlight. In Fig. 6(a),hydrated shade-grown L. pulmonaria was predarkened for36 h and then dried slowly in the dark. The desiccatedthallus was then exposed for 80 min to PPFD =1500 lmol m)2 s)1. Every 500 s a short pulse of PPFD =10 000 lmol m)2 s)1 was given to check for residual pho-tochemical activity. During illumination with 1500 lmolm)2s)1, steady-state fluorescence declined. Light pulsesproduced some additional transient quenching. Darkening
(a) (b)
Fig. 4 Effect of 20% CO2 in air on stationaryand maximum fluorescence of hydrated sun-grown (a) and shade-grown (b) Lobaria pul-
monaria. The hydrated thalli were predark-ened for 48 h to decrease zeaxanthinconcentration. Modulated light to support Fs
fluorescence had a photosynthetic photonflux density (PPFD) of 4 lmol m)2 s)1, andthe saturating light pulses had a PPFD of10 000 lmol m)2 s)1.
Fig. 5 Modulated fluorescence of sun-grown Lobaria pulmonariaafter a desiccated thallus was hydrated in 5 lM nigericin. Stronglight pulses (photosynthetic photon flux density (PPFD)= 10 000 lmol m)2 s)1) were given every 500 s. m.b., measuringbeam. For explanation, see text.
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for c. 20 min reversed only a small part of quenching.Irreversible loss of fluorescence amounted to 14% of initialfluorescence. Hydration increased fluorescence rapidly by afactor of c. 1.5. Pulse-induced fluorescence spikes appearedafter hydration, indicating charge separation in the RCs ofPSII. Fv ⁄ Fm compared with that produced by a hydratedcontrol of the same thallus which had not been subjected tolight stress revealed 25% loss of charge separation during the80 min illumination period. Loss is attributed to RCdamage suffered during light stress in the absence of water(see also Table 3).
In Fig. 6(b), a comparable experiment is shown with partof a shade-grown thallus which had been predarkened inthe hydrated state as under Fig. 6(a) but was then slowlydried in low light conditions (PPFD = 25 lmol m)2 s)1).This caused more dehydration-induced fluorescencequenching in Fig. 6(b) than in Fig. 6(a). The initial fluores-cence was therefore below the initial intensity in Fig. 6(a).Exposure for 80 min to 1500 lmol m)2 s)1 caused lessloss of steady-state fluorescence than in Fig. 6(a). Only 4%of fluorescence was irreversibly lost. Hydration increased
fluorescence rapidly by a factor of c. 2.5. Light pulsesproduced charge separation as in Fig. 6(a), but transientpostillumination quenching indicated the presence of somezeaxanthin-dependent energy dissipation. After prolongedrecovery (not shown), comparison of Fv ⁄ Fm values observedbefore and after the 80 min illumination period revealedmuch less loss of charge separation during the 80 minperiod of exposure to strong light than in the experiment ofFig. 6(a).
The experiment in Fig. 6(c) was similar to that shown inFig. 6(b), but the predarkened hydrated thallus had beenslowly dried under stronger light (PPFD = 300 lmol m)2
s)1, or c. 20% of full sunlight) which caused damage to PSIIRCs. Effects of an 80 min period of strong illumination(1500 lmol m)2 s)1) were similar to those in Fig. 6(b), inthat irreversible loss of fluorescence was distinctly belowthat seen in Fig. 6(a). Importantly, hydration increasedfluorescence but charge separation did not recover. It wasreplaced by pulse-induced reversible quenching. The samewas seen in the control, which had been slowly dried underillumination with PPFD = 300 lmol m)2 s)1 but had notbeen exposed to 80 min of light stress in the desiccatedstate. Apparently, RCs of the shade-grown hydratedthallus were damaged while they were slowly desiccated inlight of 300 lmol m)2 s)1. Damage led to reversible pulse-induced fluorescence quenching. It shows shade-adaptedL. pulmonaria to be more sensitive to light while the lichenis hydrated than after desiccation in darkness (Fig. 6a) orunder low light (Fig. 6b). The experiment of Fig. 6(c) alsoshows that desiccation-induced quenching and its reversalby hydration are independent of the functionality of PSIIRCs.
When sun-grown predarkened hydrated L. pulmonariawas slowly desiccated either in darkness or in the light andthen exposed for 80 min to a PPFD of 1500 lmol m)2 s)1,no irreversible fluorescence loss was observed during light
(a) (b) (c)
Fig. 6 Chlorophyll fluorescence of desiccatedshade-grown Lobaria pulmonaria as affectedby illumination with a photosynthetic photonflux density (PPFD) of 1500 lmol m)2 s)1
for 80 min, subsequent darkening and thenhydration (H2O) 20 min later. Strong lightpulses (PPFD = 10 000 lmol m)2 s)1) weregiven every 500 s. In (a), after predarkeningfor 36 h, L. pulmonaria was slowly dried inthe dark, while in (b) it was dried in light witha PPFD of 25 lmol m)2 s)1, and in (c) it wasdried in light with a PPFD of 300 lmolm)2 s)1. Sensitivity of recording was identicalin (a), (b) and (c).
Table 3 Loss of light-dependent charge separation in photosystemII (PSII) reaction centres (%) after 2 h of illumination of desiccatedLobaria pulmonaria with a photosynthetic photon flux density(PPFD) of 800 lmol m)2 s)1 (mercury lamp)
Shade-grown Sun-grown
Dried in darkness 61.8 ± 10.7n = 9
35.6 ± 14.5n = 7
Dried in light(PPFD = 25 lmol m)2 s)1)
33.4 ± 18.3n = 8
22.3 ± 9.3n = 10
Measurements of charge separation as Fv ⁄ Fm in hydrated samplesbefore and after exposing desiccated samples to strong light. Timeof hydration before measuring charge separation was 2 h indarkness. Values are represented as means ± SD.
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stress. Hydration increased fluorescence and pulse-inducedcharge separation in PSII RCs was comparable to thatobserved in unstressed controls. Apparently, RCs hadremained undamaged (data not shown).
In Table 3 loss of charge separation is shown after a 2 hperiod of exposure of dark-dried L. pulmonaria and of L. pul-monaria dried in low light to a PPFD of 800 lmol m)2 s)1.Damage to RCs was most severe in desiccated shade-grownL. pulmonaria which had, after prolonged predarkening,been dried in darkness. The observed sensitivity even oflight-grown desiccated L. pulmonaria to illumination wasnot seen in other experiments, which used the method ofstress application as in Fig. 6. This is perhaps the resultof using a mercury lamp for illumination in the experimentsof Table 3. The mercury lamp had more emission in theUV than the halogen lamp which was used in all otherexperiments.
Absorption changes at 800 and 950 nm and reversibleloss of fluorescence in desiccated shade-grown thalli
Very strong illumination increased absorption at 800 and950 nm reversibly both in desiccated sun-exposed and inshade-grown L. pulmonaria (see Fig. 7b,c for desiccatedshade-grown L. pulmonaria). Fluorescence remained un-changed under strong illumination in desiccated sun-exposed L. pulmonaria, but some reversible quenching ofchlorophyll fluorescence was observed in shade-grown thalli(Fig. 7a). The quantum yield of these reactions was verylow. Radicals of chlorophyll are known to absorb at800 nm, radicals of carotenoids at c. 1000 nm (Inoue et al.,1973; Holt et al., 2005). Semilog analysis of the light-dependent formation and the dark relaxation of the reac-tions revealed fast and slower phases (not shown). Thekinetics of the fast light-on and light-off phases were similaror identical both for the fluorescence changes of Fig. 7(a)and for the fast phase of the 800 nm signal in Fig. 7(b).
This suggested a common background for the 800 nm reac-tion and quencher formation. Whether the fast part of the950 nm reaction is also related to quenching is less clearbecause of considerable noise of the 950 nm signal.
Discussion
Loss of fluorescence during desiccation as indicator ofthe activation of thermal energy dissipation in L.pulmonaria
The need for protection of PSII RCs against excess lightderives from the chemical reactivity of a few chlorophyllmolecules within the RCs. Charge separation in the RCsinitiates the use of light for photosynthesis, but leads tooxidative damage if excess light is not thermally dissipated(Barber & Andersson, 1992; Aro et al., 1993). Survivalunder strong light depends on the careful control ofrogue reactions, such as the formation of chlorophyll trip-let states and the ensuing transfer of excitation energy tooxygen which results in the formation of singlet oxygen.Efficiency of control is expressed by the extent of fluores-cence quenching because chlorophyll fluorescence, energyconservation and energy dissipation are competitive pro-cesses. Extensive loss of fluorescence and of energy con-servation during desiccation and in the dry state istherefore evidence of the activation of thermal energy dis-sipation.
However, other factors also contribute to the fluores-cence decline upon desiccation. The cortex above thealgal layer in the lichen is shading the algae (Dietz et al.,2000). Desiccation increases scattering within the cortex,thereby reducing light absorption by the algae and, con-sequently, fluorescence yield (Butler, 1962). There is alsolittle doubt that shading of the algal layer below the cor-tex by melanic pigment relieves light stress on the algalphotobionts of sun-grown L. pulmonaria (Gauslaa &
(a) (b) (c)
Fig. 7 Reversible loss of chlorophyll fluores-cence (a) of desiccated shade-grown Lobariapulmonaria and reversible increase in 800and 950 nm absorption (b, c) under illumina-tion with a photosynthetic photon flux den-sity (PPFD) of 11 000 lmol m)2 s)1. Arrowsindicate onset (down arrow) and termination(up arrow) of illumination. In (a), loss of fluo-rescence F (%) is based on the fluorescenceintensity of a desiccated thallus.
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Solhaug, 2001, 2004; Solhaug et al., 2003; McEvoyet al., 2007). Nevertheless, only a little fluorescence waslost during desiccation after a thallus had been treatedwith glutaraldehyde, an inhibitor of desiccation-inducedfluorescence quenching (Fig. 2). Therefore, we concludethat increased attenuance in the cortex only contributesto the fluorescence loss in desiccated L. pulmonaria to aminor extent. Most of this effect is the result of fluores-cence quenching by activation of thermal energy dissipa-tion. Similar conclusions, based on fluorescence lifetimemeasurements, were drawn by Veerman et al. (2007) forthe fluorescence decrease during desiccation of Parmeliasulcata.
Molecular basis of desiccation-induced energydissipation
Previous investigations (Heber et al., 2007; Heber, 2008)have led to the conclusion that desiccation-induced fluores-cence quenching results from desiccation-induced confor-mational changes of a chlorophyll protein. This conclusionwas based on different observations: fast drying in darknessled to less fluorescence quenching than slow drying; fastdrying decreased charge separation in PSII RCs less thanslow drying; heating (intended to inactivate chlorophyllproteins by unfolding) increased fluorescence of rapidlydried desiccation-tolerant photoautotrophs less than that ofslowly dried photoautotrophs; and glutaraldehyde inhibiteddesiccation-induced fluorescence quenching.
The present work with L. pulmonaria confirms andextends these observations. Strong desiccation-induced fluo-rescence quenching was also observed in L. pulmonaria. Itsextent differed between shade- and sun-grown thalli(Fig. 1). Loss of fluorescence during desiccation wasreversed when the desiccated thalli were heated (Fig. 3).Also here, different responses were observed for shade- andsun-grown thalli. Glutaraldehyde inhibited desiccation-induced quenching (Fig. 2).
In all lichens investigated so far, desiccation inducedstrong fluorescence quenching. This occurs not only inchlorolichens, which possess a chlorophyll b containinglight harvesting complex, but also in the cyanolichen Pelti-gera neckeri, which does not (Heber et al., 2007). It there-fore appears that the chlorophyll protein complex whichchanges its conformation upon drying is located within orclose to the core of PSII, not in the antenna system. In cy-anobacteria, c. 30 chlorophyll molecules reside in the coreof PSII. They are in, or are close to, excitation equilibriumwith the RCs of PSII (Mimuro & Kikuchi, 2003).
In lichens and desiccation-tolerant mosses, preferentialquenching of the main emission of PSII fluorescence at c.685 nm during desiccation is accompanied by far-red emis-sion which is attributable to PSII (Heber & Shuvalov,2005). This has led to the suggestion that a long-wavelength
emitter coupled to PSII is involved in desiccation-inducedfluorescence quenching. Strong support for this came fromrecent measurements of fluorescence life times (Veermanet al., 2007). The excitation life time of the far-red emitterwas dramatically shortened by desiccation of the lichen P.sulcata. De-excitation of the bulk pool of excited chloro-phyll was eight times faster after desiccation than in thehydrated state (Veerman et al., 2007). This drains excita-tion energy from the RCs, protecting them against photoin-activation.
Photoreactions were readily observable in desiccatedshade-grown thalli as reversible photo-induced absorptionchanges at 800 and 950 nm. These changes were accompa-nied by kinetically closely related quenching of chlorophyllfluorescence (Fig. 7). Oxidation of chlorophylls is knownto result in increased absorption at 800 nm. Reactivechlorophylls reside in the RCs of PSI and PSII. The RC ofPSII contains six chlorophylls and two beta-carotenes.Light-dependent and reversible oxidation of a carotene hasbeen observed in desiccated leaf fractions, which, in contrastto lichens, are accessible to transmission spectrophotometricanalysis in the visible range (Shuvalov & Heber, 2003). ForL. pulmonaria, Fig. 7(c) shows light-dependent absorptionchanges at 950 nm where carotenoid cations absorb (Holtet al., 2005). When oxidized, beta-carotene takes anelectron from a neighbouring chlorophyll, ChlZD2. Thisproduces a quencher (Faller et al., 2006). The similarity ofthe fast responses of fluorescence and of the 800 nmabsorption change shown in Fig. 7(a,b) suggests formationof a quencher in the RC of PSII which could contributeto photoprotection by permitting energy dissipationwithin the RC. Significantly, the fast phase of the lightreactions seen in photosensitive, desiccated, shade-grownL. pulmonaria as fast changes in 800 and 950 nmabsorption was small or absent in the more phototolerantsun-grown L. pulmonaria.
The effect of light on desiccation-induced quenching
Low light, when present during desiccation, increased fluo-rescence quenching, heat-induced reversal of fluorescencequenching and phototolerance (Figs 1 and 3, Table 3).This was true both for the more photosensitive shade-grownand the more phototolerant sun-grown L. pulmonaria. Theeffect of the presence of light during drying has also beenobserved with other chlorolichens such as C. rangiformisand P. sulcata (Heber et al., 2007; Heber, 2008). ForL. pulmonaria, it was recently reported by Stepigova et al.(2008).
How can the effect of light on desiccation-inducedfluorescence quenching be explained? In hydrated higherplants, light controls the thermal dissipation of excess lightenergy that cannot be used for photosynthesis (Niyogi,1999; Holt et al., 2004; Ruban et al., 2007). Necessary for
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activation of energy dissipation are the presence of thexanthophyll zeaxanthin (Demmig-Adams, 1990; Bjorkman& Demmig-Adams, 1994; Ahn et al., 2008) and thelight-dependent protonation of a thylakoid protein, thePsbS-protein (Li et al., 2004; Takizawa et al., 2007).However, the protonophore nigericin which dissipates theDpH across the thylakoid membrane and inhibits thesynthesis of zeaxanthin in the light did not inhibit desicca-tion-induced quenching in L. pulmonaria. The experimentof Fig. 5 shows that inhibition of zeaxanthin synthesis andof protonation of the PsbS protein by nigericin had little orno effect on the extent of quenching of fluorescence duringdesiccation of sun-grown L. pulmonaria.
Apparently, light is capable of interacting with the con-formational changes of a chlorophyll protein which areinduced by desiccation. Interaction could involve increasedbinding of zeaxanthin or other carotenoids. Table 2 showsthat zeaxanthin was present in the thalli even after pro-longed predarkening and desiccation of the thalli in thedark.
Sensitivity of desiccated L. pulmonaria to strong light
Gauslaa & Solhaug (1999, 2004, 2001) have reportedlight-induced damage to desiccated thalli of L. pulmonaria.Their observations are confirmed by the experiments shownin Fig. 6 and in Table 3, where partial loss of functionalityof RCs after prolonged illumination of desiccated thalli,with light not even reaching full sunlight, is documented.More damage was inflicted to shade-grown than tosun-grown. L. pulmonaria. Also, thalli that had been driedin darkness were more damaged than those dried in thelight, confirming results obtained by Stepigova et al.(2008).
Fluorescence was less quenched after desiccation of L.pulmonaria in darkness than after desiccation in the pres-ence of light. Only a little light was required for increasedfluorescence quenching during desiccation. Also, shade-grown thalli displayed less desiccation-induced quenchingthan sun-grown thalli. A negative correlation between lightdamage and fluorescence quenching is in line with thehypothesis that the latter is indeed photoprotective. Onemay argue that photodamage to L. pulmonaria is notentirely absent in desiccated thalli, pointing out that protec-tion by desiccation does not exclude damage. However,other lichens, such as P. sulcata or Hypogymnia physodes, aremore resistant to photoinhibition in the desiccated statethan L. pulmonaria. Considering the sensitivity to lightwhile water is still present (as shown by the almost total lossof PSII charge separation after slow desiccation in a PPFDof 300 lmol m)2 s)1 and ensuing illumination in the drystate (Fig. 6c)), the protection indicated by strong fluores-cence quenching in the dry state is still dramatic, also in L.pulmonaria.
Adjustment of desiccation-induced energy dissipationto the irradiance
On desiccation, fluorescence was more quenched in sun-grown than in shade-grown L. pulmonaria (Table 1). Itappears that photoprotection is regulated in L. pulmonariaboth in the long term by growth and exposure conditionsand in the much shorter term by the presence or absence oflight during desiccation. Prolonged exposure to strong lightduring growth leads to slow melanin formation by the my-cobiont as a sunscreen (Gauslaa & Solhaug, 2001) but alsopermits increased fluorescence quenching during desiccation(Fig. 1, Table 1). The molecular basis for increased fluores-cence quenching in the sun-grown thalli is still unclear, but amain factor could be increased synthesis of desiccation-responsive chlorophyll protein. In the shorter term, avail-ability of light during desiccation increases fluorescencequenching and, thereby, phototolerance (Table 3), probablyby increased dissipative interaction between pigments withinthe desiccation-responsive protein. The interplay of thesefactors permits considerable flexibility in the adaptation tochanging light stress conditions in the different seasons of ayear as well as acclimation to habitats with different lightclimates (Gauslaa & Solhaug, 2000; Gauslaa et al., 2005).
Acknowledgements
UH wishes to acknowledge long-standing cooperation withAkademik V. A. Shuvalov, Russian Academy of Sciences,Pushchino-na-Oke, which helped to shape his views on therelationship between energy conservation and energy dissi-pation in photosynthesis. Professor R. Hedrich providedlaboratory space and facilities at the University of Wurz-burg. We thank Dr. U. Schreiber, Dr. C. Klughammer andU. Schliwa for technical help. We are grateful to reviewersof a first submission of our work for their criticism. MareikeJezek is thanked for skilful and patient help with thepreparation of the figures.
