Photoprotection of reaction centres in photosynthetic organisms: mechanisms of thermal energy dissipation in desiccated thalli of the lichen Lobaria pulmonaria


Author for correspondence:
Wolfgang Bilger
Tel: +49 431 880 4289


  • 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 activation 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 (F0). Sun-grown thalli, as well as thalli exposed to low irradiance during drying, showed a larger reduction of F0 than shade-grown thalli or thalli desiccated in the dark. Desiccation increased phototolerance, which was positively correlated to enhanced quenching of F0. Quenching of F0 could be reversed by heating, and could be inhibited by glutaraldehyde but not by the uncoupler nigericin.
  • Activation of energy dissipation, apparent as F0 quenching, is proposed to be based on an alteration in the conformation of a pigment protein complex. This permits thermal energy dissipation and gives considerable flexibility to photoprotection. 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.

minimum fluorescence, QA in the RC of PSII oxidized


maximum fluorescence, QA reduced


stationary fluorescence, QA partially reduced


variable fluorescence, Fm′-Fs or Fm-F0


nonphotochemical fluorescence quenching, Fm/Fm′– 1 or, in desiccated thalli, Fm/F0 desiccated − 1 because Fm is very close to F0 desiccated


photosynthetic photon flux density


photosystem I


photosystem II


quinone acceptor in the reaction centre of PSII


reaction centre


Many lichens are desiccation-resistant as they survive periods of total dehydration (see Nash, 2008). Moreover, in contrast to hydrated and metabolically active lichens, most dry lichens tolerate long-lasting high incident light intensities 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 daily high light intensities. Recent experiments have shown that lichens are even able to survive dryness, heat or cold, and incident radiation for 14 d when openly exposed to the extraterrestrial space (Sancho et al., 2007). Thus, resistance to light of such species seems to be rather unlimited in principle. However, Gauslaa & Solhaug (1999, 2001) also demonstrated that light can damage lichens while they are dry. This leads to the conclusion that high photoresistance of dry lichens is not a general feature of thallus dehydration. Rather, specific mechanisms must be involved to facilitate protection against excessive light. In the present contribution, which is a continuation of previous work (Kopecky et al., 2005; Heber et al., 2006a,b, 2007; Heber, 2008), we investigate such mechanisms. As before, we use fluorescence responses to desiccation and hydration in order to obtain information on energy dissipation. We compare thalli of the foliose lichen Lobaria pulmonaria that have grown in the deep shade with those that are adapted to full sunlight, because it is known that differences in growth conditions result in differences in phototolerance. L. pulmonaria is especially suited for our experiments because much information about its performance has been obtained by Gauslaa and his group and is available in the literature (Gauslaa & Solhaug, 1999, 2000, 2001; McEvoy et al., 2007; Stepigova et al., 2008). L. pulmonaria uses a xanthophyll cycle to synthesize zeaxanthin in the light. Zeaxanthin plays a central role in the photoprotection of many plants (Demmig-Adams, 1990). In our work, we have tried to answer the following questions. Can we explain differences in phototolerance between thalli from sun-exposed and thalli from deeply shaded habitats? What are the mechanisms that contribute to photoprotection? How are they activated? How important is the well-investigated mechanism of zeaxanthin-dependent energy 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 of Acer pseudoplatanus at Ramsau, Germany, not far from Berchtesgaden, at an altitude of 800 m. This tree is exposed to the sun during all seasons; the lichen specimens were collected from the side exposed to the south/south-west. Thalli grown in the deep shade were collected from the bark of willow shrubs (Salix sp.) to the south of the Hintersee lake at an altitude of 800 m. The specimens grew 60 cm above the soil in the deep shade of the closed canopy of Salix sp. and Picea abies. The thalli from the sun-exposed site were distinctly brownish, whereas the shade-grown samples were greyish-green. Dates of collection were 27 July 2005 and 26 January 2008. The samples were sent to Würzburg in the desiccated state, where they were stored at −20°C. No deterioration of fluorescence signals was observed over the storage period. Experiments in Figs 1, 4 and 7 were done in March and November 2008 with the samples from 2008; the remaining experiments were done during November 2007 with the samples from 2005.

Figure 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 weak light 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 μmol m−2 s−1) given during the first and third hydration cycle are recognizable by large transient fluorescence responses. They were intended to indicate the maximal variable fluorescence Fv = (Fm − F0). Actinic light was absent and light pulses were weak (PPFD = 2 μmol m−2 s−1, recognizable by small fluorescence spikes) during most of the first and third hydration cycles. Actinic light was present (PPFD = 300 μmol m−2 s−1) during most of the second hydration cycle where all light pulses given were strong. Sensitivity of fluorescence recording was identical in (a) and (b) so that fluorescence intensities can be compared. m.b., measuring beam. For further explanation see text.

Prolonged dark adaptation (36 or 48 h) of hydrated thalli was intended to minimize zeaxanthin concentrations. Zeaxanthin is known to be converted to violaxanthin in the dark or in low light (Hager, 1980; Björkman & Demmig-Adams, 1994). The thalli were slowly dried in the dark at room 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 an average photosynthetically active photon flux density (PPFD) of 0.04 μmol m−2 s−1 was present during slow drying to enable the recording of fluorescence emission. Absence of activity of zeaxanthin-dependent energy dissipation in predarkened hydrated thalli was checked by making sure that quenching of basal or F0 chlorophyll fluorescence was not induced by illumination with strong light pulses which lasted 1 s (Katona et al., 1992; Kopecky et al., 2005; Heber et al., 2006b). Active zeaxanthin-dependent energy dissipation is indicated by a short and transient postillumination F0 quenching response which was carefully distinguished from postillumination oxidation of reduced QA (quinone acceptor) in reaction centres (RCs) of photosystem II (PSII).

Modulated chlorophyll fluorescence was measured beyond 700 nm after excitation at c. 650 nm (using the far-red transmitting filter RG 9; Schott, Mainz, Germany) by a pulse amplitude modulation fluorometer (PAM 101; Walz, Effeltrich, Germany) (Schreiber et al., 1986).

Short pulses (usually 1 s) of white light (Calflex c and DT-Cyan filters; Balzers, Liechtenstein) from a halogen lamp (KL 1500; Schott) were brought to the cuvette by fibreoptics to probe for variable fluorescence. The PPFD of the light pulses was usually c. 10 000 μmol m−2 s−1 (but only 2 or 4 μmol m−2 s−1 when high light fluxes had to be avoided). Actinic light was provided by a second halogen lamp. Heat- and far-red-absorbing filters were a combination of Calflex c and DT-Cyan. For light stress experiments with desiccated thalli lasting > 30 min, strong light from either a halogen or a mercury lamp was used (PPFD ≤ 1500 μmol m−2 s−1). At comparable PPFDs (as measured by a Li-Cor 189 quantum sensor; Walz), the mercury lamp (Osram Powerstar HQI-R, 250 W; Osram GmbH, München, Germany) produced more photodamage than the halogen lamp. Whenever necessary, the temperature of the samples was monitored by a thermocouple. Heating experiments and experiments with gas mixtures of different CO2 content were performed using a sandwich-type cuvette which permitted controlled gas flow over the thalli.

Light-dependent absorption changes at 800 nm were measured in reflection using the PAM fluorometer in combination with an ED800 T emitter/detector unit (Walz). This attachment was modified for the measurement of absorption changes at 950 nm by replacing the original LED of the emitter/detector unit with an LED with peak emission at 950 nm.

For carotenoid determinations, desiccated L. pulmonaria thalli were stored in a freezer at −20°C. Before extraction, they were washed twice with 100% acetone, subsequently briefly rewetted in 20 mM Hepes solution in darkness and then ground in liquid nitrogen in a mortar before adding 80% acetone/20% 20 mM Hepes. After centrifugation, pellets were extracted twice with 100% acetone. Combined extracts were analysed for carotenoids and chlorophyll a and b using an Agilent 1100 HPLC system with diode array detection at 445 and 407 nm following essentially the method described in Niinemets et al. (1998).

Glutaraldehyde was obtained from Sigma-Aldrich/Fluka (Seelze, Germany).


Changes in emission of chlorophyll fluorescence during desiccation and hydration

Lichens respond to desiccation and hydration by large changes in fluorescence emission (Lange et al., 1989). Figure 1 shows such changes for thalli of L. pulmonaria in both hydrated and desiccated states. The thalli had been collected in late January under alpine winter conditions. On addition of water to desiccated thalli, chlorophyll fluorescence increased more in sun-exposed than in shade-grown thalli. Slow drying after hydration resulted in greater loss of fluorescence in sun-exposed than in shade-grown thalli. When not caused by altered light absorption, loss of fluorescence indicates activation of energy dissipation because fluorescence and thermal energy dissipation compete directly for the energy of absorbed light when photochemical use of light energy is negligible, as is the case in the absence of water. Greater desiccation-induced fluorescence quenching is equivalent to greater thermal dissipation of absorbed light energy. Greater loss of fluorescence upon desiccation of sun-grown vs shade-grown L. pulmonaria therefore suggests better photoprotection of sun-grown than of shade-grown L. pulmonaria.

The extent of the increase of fluorescence observed on hydration depended on the previous history of the thalli. In the experiment illustrated in Fig. 1(a), basal fluorescence, F0′, of sun-grown desiccated L. pulmonaria increased on hydration to the value termed F0. The ratio F0/F0′ was 7.7. After slow drying in near darkness (the very weak measuring beam needed for the measurement of fluorescence had a PPFD of only 0.04 μmol m−2 s−1), the ratio F0/F0′ decreased to 4.8 (Table 1). Nonphotochemical fluorescence quenching (NPQ) had been 9 before drying and was 5.5 afterwards. However, after the same thallus was hydrated a second time, and then dried slowly in light of PPFD = 300 μmol m−2 s−1, which is c. 20% of full sunlight, F0′ was lowered to F0′′′. The ratio F0/F0′′′ was now 12.9, and NPQ was 15. A third hydration returned fluorescence almost to the initial F0 intensity. Slow drying under the same conditions of near darkness as during the first drying returned the ratio F0/F0′′′′ to the previous ‘dark’ value of 4.8. In other words, drying the thallus in near darkness did not suppress F0 as much as it did during drying in the presence of illumination. Not much illumination during slow drying was needed to observe this effect. It was similar with PPFDs of 300, 130 and 25 μmol m−2 s−1 (data not shown). Increased fluorescence quenching after desiccation of L. pulmonaria in the light has recently also been observed by Stepigova et al. (2008).

Table 1.   Ratios F0 hydrated/F0 desiccated in the experiments of Fig. 1
F0/F0′′, dark-dried, after first hydration4.82.5
F0/F0′′′, light-dried, after second hydration12.93.7
F0/F0′′′′, dark-dried, after third hydration4.82.7

Strong 1 s light pulses of PPFD = 11 000 μmol m−2 s−1 (equivalent to more than six times full sunlight) failed to change fluorescence appreciably when the thalli were desiccated. Similar observations were made before with different algal and cyanobacterial lichens (Lange et al., 1989). After a few minutes’ hydration, strong pulses increased fluorescence to the Fm value. Fv/Fm was 0.26 shortly after the first hydration, that is, less than the maximum quantum efficiency commonly observed in predarkened leaves, where Fv/Fm ratios are c. 0.8 (Björkman & Demmig, 1987), and also lower than the values 0.63–0.76 that are usually found after prolonged dark adaptation in lichen photobionts (Jensen, 2002). Transient postillumination F0 quenching immediately after strong light pulses indicated the induction of zeaxanthin-dependent energy dissipation by the light pulses (Katona et al., 1992; Heber et al., 2007). This effect disappeared during the experiment as shown by the absence of postillumination F0 quenching when a strong light pulse was given later on. Charge separation in PSII RCs was still significant after strong light pulses were replaced by very weak light pulses of PPFD = 2 μmol m−2 s−1. Under the weak pulses, fluorescence responses were much reduced. As drying progressed, charge separation decreased until it was no longer noticeable.

The second hydration increased fluorescence and re-established charge separation but the strong light pulses failed to be followed by postillumination F0 quenching this time, showing that zeaxanthin-dependent energy dissipation had been slowly inactivated under the near-darkness conditions of the first hydration. When continuous illumination with PPFD = 300 μmol m−2 s−1 was added together with the strong light pulses, the transient fluorescence peaks elicited by the light pulses decreased to Fm′. The lowering of Fm to Fm′ is attributed to nonphotochemical fluorescence quenching. The purpose of illumination had been to activate zeaxanthin-dependent energy dissipation. In the presence of actinic illumination, some RCs were closed and did not contribute to the fast pulse-induced fluorescence responses. As water continued to be lost, not only stationary fluorescence but also pulse-induced fluorescence responses decreased. The final fluorescence intensity after drying had been complete was below that observed after the first hydration. Apparently, illumination during hydration had increased fluorescence quenching. This confirms observations reported in a recent publication of Stepigova et al. (2008).

In principle, the third hydration returned the thallus to the situation observed during the first hydration except for one important point. Immediately after the first light pulses, small transient postillumination quenching effects were observed which were absent after the first hydration. Apparently, zeaxanthin-dependent energy dissipation had been activated during the second hydration. Postillumination F0 quenching slowly disappeared during hydration as shown by its absence when a strong light pulse were given later on during the hydration phase. It seems that zeaxanthin-dependent energy dissipation was slowly lost under the near-darkness condition of the third hydration. The same had been observed during the first hydration.

Figure 1(b) shows, for desiccated shade-grown L. pulmonaria, the same procedure described for sun-grown L. pulmonaria in Fig. 1(a). The sensitivity of measurements was identical in Figs 1(a) and (b). Three differences need to be emphasized, as follows. First, F0′ fluorescence intensities were higher in shade-grown than in sun-adapted L. pulmonaria, but ratios F0/F0′ were lower (see Table 1); NPQ values were lower than in sun-grown L. pulmonaria, suggesting there was less desiccation-induced energy dissipation. Secondly, continuous illumination during the second hydration decreased pulse-induced fluorescence responses more, but stationary fluorescence less, than in sun-grown L. pulmonaria. And thirdly, during the third hydration, quantum efficiencies of charge separation as expressed by Fv/Fm values were 12% below the values measured during the first hydration. This decline suggested damage to the RCs while the shade-grown L. pulmonaria had received a PPFD of 300 μmol m−2 s−1 during the second hydration phase.

Table 1 compares the ratios F0 hydrated/F0 desiccated observed in the experiment whose results are shown in Fig. 1. Desiccation-induced quenching of fluorescence was stronger in sun-grown than in shade-grown L. pulmonaria, whether or not the thalli were dried in darkness or in the light. The presence of light during desiccation increased the F0 hydrated/F0 desiccated ratios.

Inhibition of desiccation-induced loss of fluorescence by glutaraldehyde

The amplitude of chlorophyll fluorescence is a product of light absorption by photosynthetic pigments and the quantum yield of fluorescence emission. The latter is decreased by the activation of a competitive reaction such as nonradiative energy dissipation. Desiccation of various lichens, and in particular of L. pulmonaria (Gauslaa & Solhaug, 2001), has been reported to decrease light transmission to the algal layer (Dietz et al., 2000; Heber et al., 2007). There was a question as to what extent this affects the desiccation-induced fluorescence quenching shown in Fig. 1. Glutaraldehyde has been shown to inhibit desiccation-induced fluorescence quenching in chlorolichens such as Parmelia sulcata and Cladonia rangiformis (Heber et al., 2007; Heber, 2008). It possesses two reactive aldehyde groups capable of reacting with proteins but does not alter light absorption by pigments. Figure 2 shows emission of modulated chlorophyll 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 μmol m−2 s−1. Strong light pulses given every 500 s increased fluorescence transiently, thereby revealing residual light-dependent charge separation. During progressive loss of water, fluorescence decreased and pulse-induced fluorescence responses changed direction as desiccation neared completion. Fluorescence emission from desiccated glutaraldehyde-treated thalli was stronger than fluorescence emission from desiccated thalli 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). This compares with the much larger F0 hydrated/F0 desiccated ratios listed in Table 1. The loss of fluorescence still observed in Fig. 2 after desiccation is presumably attributable either to incomplete inhibition of fluorescence quenching by glutaraldehyde or to loss of light absorption during desiccation, or a combination of both.

Figure 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 photosynthetic photon flux density (PPFD) of 4 μmol m−2 s−1. Very strong light pulses were given every 500 s to probe for charge separation in photosystem II (PSII) reaction centres (RCs). Note the reversal of direction of pulse-induced fluorescence responses during drying. m.b., measuring beam.

Heat-induced increase of fluorescence emission in desiccated thalli

Recently, it was suggested that desiccation of lichens changed the conformation of a pigment protein complex so as to create dissipating centres within the complex (Heber et al., 2007; Heber, 2008). Heating inactivates proteins by unfolding secondary and tertiary protein structures. When the desiccated lichen P. sulcata was heated, chlorophyll fluorescence increased and charge separation in PSII RCs was re-established as indicated by the appearance of pulse-induced fluorescence spikes (Heber & Shuvalov, 2005).

Figure 3 shows that increasing the temperature of desiccated L. pulmonaria thalli decreased fluorescence slightly between 10 and 30°C before it increased strongly above 60°C until no further increase was observed close to 80°C. Importantly, the heat-induced fluorescence increase was larger 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 and light-dried thallus (Fig. 3b), which had lost more fluorescence on desiccation than a thallus dried in the dark (Fig. 1a). The data suggest that the desiccation-induced quenching of chlorophyll fluorescence can be reversed by heating desiccated thalli. However, in contrast to previous observations with P. sulcata (Heber & Shuvalov, 2005), variable fluorescence could not be restored upon heating of L. pulmonaria. Only some photochemical activity appeared at high temperature, as indicated by the reversible fluorescence quenching induced by strong light pulses (Fig. 3). The difference between P. sulcata and L. pulmonaria was probably the result of the higher heat sensitivity of the latter (Gauslaa & Solhaug, 1999).

Figure 3.

 Changes of fluorescence of desiccated sun-grown (a, b) and shade-grown (c, d) Lobaria pulmonaria during heating and cooling. Heating rate was c. 2°C min−1 and the actual temperature (°C) is indicated on top of the fluorescence trace. Hydrated thalli were predarkened 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 μmol m−2 s−1 (b, d). Measurements were done in darkness, and strong light pulses were given every 200 s.

Zeaxanthin cycle and zeaxanthin-dependent energy dissipation in L. pulmonaria

Increased desiccation-induced quenching under illumination suggested a role of zeaxanthin in energy dissipation because zeaxanthin is known to be synthesized in the light from violaxanthin (Demmig-Adams, 1990). When present, it contributes to photoprotection under the control of the PsbS protein of the thylakoids (Li et al., 2004; Takizawa et al., 2007). Components of the xanthophyll cycle (zeaxanthin, violaxanthin and antheraxanthin) were measured in desiccated thalli that had been predarkened in the hydrated state for 36 h and were then slowly desiccated either in darkness or in light of PPFD = 300 μmol m−2 s−1. Results are shown in Table 2. They reveal that drying of hydrated sun-grown L. pulmonaria in the light increased concentrations of zeaxanthin at the expense of violaxanthin. Zeaxanthin decreased after drying in the dark, whereas violaxanthin increased. Very similar observations were made with shade-grown L. pulmonaria, but zeaxanthin increased less during drying in the light than in sun-grown L. pulmonaria. Even predarkening of hydrated L. pulmonaria for 36 h did not eliminate zeaxanthin fully.

Table 2.   Antheraxanthin (A), zeaxanthin (Z), violaxanthin (V) and the sum of antheraxanthin + zeaxanthin + violaxanthin (VAZ) in desiccated Lobaria pulmonaria (thalli collected in summer)
  1. Contents in mmol mol−1 chlorophyll; means of five determinations ± SD.

  2. 1Photosynthetic photon flux density (PPFD) = 300 μmol m−2 s−1.

L. pulmonaria, grown in the sun
 Dried in the light1113 ± 718 .1 ± 3.218.1 ± 0.7149 ± 3.5
 Dried in darkness21.4 ± 3.515.2 ± 4.787.2 ± 11.3124 ± 11.8
L. pulmonaria, grown in the shade
 Dried in the light147 ± 11.913.6 ± 4.722.6 ± 2.283.3 ± 14.2
 Dried in darkness28.3 ± 15.611.8 ± 2.954.3 ± 4.594.3 ± 17.4

The data show functioning of the xanthophyll cycle but do not prove that activation of zeaxanthin-dependent energy dissipation by light during hydration is necessary for photoprotection 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 responsible for activating zeaxanthin-dependent energy dissipation, 20% CO2 in air, acting as a potential acid, has also been shown to be effective in activating energy dissipation in lichens and mosses when zeaxanthin is present but light is absent (Bukhov et al., 2001). No quenching was observed in the absence of zeaxanthin. Figure 4 shows that even after 48 h predarkening, considerable quenching of fluorescence was produced by 20% CO2 in hydrated L. pulmonaria. Quenching was stronger in sun-grown predarkened thalli than in shade-grown thalli. The ratio of stationary fluorescence 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 was caused by postillumination oxidation of reduced QA, not by F0 quenching. The experiments not only confirmed the presence of zeaxanthin in the thalli even after prolonged predarkening but also the necessity of protonation for the activation of zeaxanthin-dependent energy dissipation. Moreover, they showed persistence of light-dependent charge separation in the presence of energy dissipation activated by CO2. The ratio (Fm− F)/Fm, which serves to indicate charge separation in PSII RCs, decreased under the influence of 20% CO2 by 25% in shade-grown L. pulmonaria and by 35% in sun-grown L. pulmonaria. CO2-induced NPQ 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).

Figure 4.

 Effect of 20% CO2 in air on stationary and maximum fluorescence of hydrated sun-grown (a) and shade-grown (b) Lobaria pulmonaria. The hydrated thalli were predarkened for 48 h to decrease zeaxanthin concentration. Modulated light to support Fs fluorescence had a photosynthetic photon flux density (PPFD) of 4 μmol m−2 s−1, and the saturating light pulses had a PPFD of 10 000 μmol m−2 s−1.

To answer the question as to whether activation of zeaxanthin-dependent energy dissipation is responsible for the light effect on fluorescence quenching during desiccation, as shown in Fig. 1, attempts were made in the experiment of Fig. 5 to interfere with the control of zeaxanthin-dependent energy dissipation by the PsbS protein. Protonation of the PsbS protein requires a lowering of the intrathylakoid pH by proton-coupled light-dependent electron transport. This can be prevented by the protonophore nigericin, which is also an effective inhibitor of zeaxanthin synthesis in the light. In the experiment of Fig. 5, part of a sun-grown hydrated thallus was predarkened for 36 h to decrease zeaxanthin concentrations (see Table 2). It was then slowly dried in the dark and subsequently hydrated in 5 μM nigericin. Effectiveness of the inhibition of intrathylakoid proton deposition by nigericin was demonstrated by the sensitive response of chlorophyll fluorescence to low light (PPFD = 2 μmol m−2 s−1) which raised fluorescence transiently to the Fm value in the presence of nigericin, but not in its absence (see Fig. 1). This indicates inhibition of electron transport to a large extent, probably because of blockage of photosynthesis as a result of inhibition of photophosphorylation. Slow drying of the nigericin-treated hydrated thallus under illumination with PPFD = 300 μmol m−2 s−1 quenched fluorescence as much as it did in an untreated illuminated control thallus and more than in a predarkened thallus which had been dried in near darkness. The ratio F0/F0′ was 9.1 in the experiment of Fig. 5 and the ratio Fm/F0′′ 14.8. The corresponding ratios in a control experiment without nicericin were 8.7 and 16.3. A nigericin experiment similar to that shown in Fig. 5 was also performed with the chlorolichen C. rangiformis (Heber, 2008). The results were similar to those of the L. pulmonaria experiment of Fig. 5.

Figure 5.

 Modulated fluorescence of sun-grown Lobaria pulmonaria after a desiccated thallus was hydrated in 5 μM nigericin. Strong light pulses (photosynthetic photon flux density (PPFD) = 10 000 μmol m−2 s−1) were given every 500 s. m.b., measuring beam. For explanation, see text.

Irreversible loss of fluorescence and of light-dependent charge separation in desiccated L. pulmonaria under prolonged strong illumination

Figure 6 shows representative examples of light stress experiments. They are intended to give information on the sensitivity of desiccated L. pulmonaria to sunlight. In Fig. 6(a), hydrated shade-grown L. pulmonaria was predarkened for 36 h and then dried slowly in the dark. The desiccated thallus was then exposed for 80 min to PPFD = 1500 μmol m−2 s−1. Every 500 s a short pulse of PPFD = 10 000 μmol m−2 s−1 was given to check for residual photochemical activity. During illumination with 1500 μmol m−2s−1, steady-state fluorescence declined. Light pulses produced some additional transient quenching. Darkening for c. 20 min reversed only a small part of quenching. Irreversible loss of fluorescence amounted to 14% of initial fluorescence. Hydration increased fluorescence rapidly by a factor of c. 1.5. Pulse-induced fluorescence spikes appeared after hydration, indicating charge separation in the RCs of PSII. Fv/Fm compared with that produced by a hydrated control of the same thallus which had not been subjected to light stress revealed 25% loss of charge separation during the 80 min illumination period. Loss is attributed to RC damage suffered during light stress in the absence of water (see also Table 3).

Figure 6.

 Chlorophyll fluorescence of desiccated shade-grown Lobaria pulmonaria as affected by illumination with a photosynthetic photon flux density (PPFD) of 1500 μmol m−2 s−1 for 80 min, subsequent darkening and then hydration (H2O) 20 min later. Strong light pulses (PPFD = 10 000 μmol m−2 s−1) were given every 500 s. In (a), after predarkening for 36 h, L. pulmonaria was slowly dried in the dark, while in (b) it was dried in light with a PPFD of 25 μmol m−2 s−1, and in (c) it was dried in light with a PPFD of 300 μmol m−2 s−1. Sensitivity of recording was identical in (a), (b) and (c).

Table 3.   Loss of light-dependent charge separation in photosystem II (PSII) reaction centres (%) after 2 h of illumination of desiccated Lobaria pulmonaria with a photosynthetic photon flux density (PPFD) of 800 μmol m−2 s−1 (mercury lamp)
  1. Measurements of charge separation as Fv/Fm in hydrated samples before and after exposing desiccated samples to strong light. Time of hydration before measuring charge separation was 2 h in darkness. Values are represented as means ± SD.

Dried in darkness61.8 ± 10.7
= 9
35.6 ± 14.5
= 7
Dried in light (PPFD = 25 μmol m−2 s−1)33.4 ± 18.3
= 8
22.3 ± 9.3
= 10

In Fig. 6(b), a comparable experiment is shown with part of a shade-grown thallus which had been predarkened in the hydrated state as under Fig. 6(a) but was then slowly dried in low light conditions (PPFD = 25 μmol m−2 s−1). This caused more dehydration-induced fluorescence quenching in Fig. 6(b) than in Fig. 6(a). The initial fluorescence was therefore below the initial intensity in Fig. 6(a). Exposure for 80 min to 1500 μmol m−2 s−1 caused less loss 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 pulses produced charge separation as in Fig. 6(a), but transient postillumination quenching indicated the presence of some zeaxanthin-dependent energy dissipation. After prolonged recovery (not shown), comparison of Fv/Fm values observed before and after the 80 min illumination period revealed much less loss of charge separation during the 80 min period of exposure to strong light than in the experiment of Fig. 6(a).

The experiment in Fig. 6(c) was similar to that shown in Fig. 6(b), but the predarkened hydrated thallus had been slowly dried under stronger light (PPFD = 300 μmol m−2 s−1, or c. 20% of full sunlight) which caused damage to PSII RCs. Effects of an 80 min period of strong illumination (1500 μmol m−2 s−1) were similar to those in Fig. 6(b), in that irreversible loss of fluorescence was distinctly below that seen in Fig. 6(a). Importantly, hydration increased fluorescence but charge separation did not recover. It was replaced by pulse-induced reversible quenching. The same was seen in the control, which had been slowly dried under illumination with PPFD = 300 μmol m−2 s−1 but had not been exposed to 80 min of light stress in the desiccated state. Apparently, RCs of the shade-grown hydrated thallus were damaged while they were slowly desiccated in light of 300 μmol m−2 s−1. Damage led to reversible pulse-induced fluorescence quenching. It shows shade-adapted L. pulmonaria to be more sensitive to light while the lichen is hydrated than after desiccation in darkness (Fig. 6a) or under low light (Fig. 6b). The experiment of Fig. 6(c) also shows that desiccation-induced quenching and its reversal by hydration are independent of the functionality of PSII RCs.

When sun-grown predarkened hydrated L. pulmonaria was slowly desiccated either in darkness or in the light and then exposed for 80 min to a PPFD of 1500 μmol m−2 s−1, no irreversible fluorescence loss was observed during light stress. Hydration increased fluorescence and pulse-induced charge separation in PSII RCs was comparable to that observed in unstressed controls. Apparently, RCs had remained undamaged (data not shown).

In Table 3 loss of charge separation is shown after a 2 h period of exposure of dark-dried L. pulmonaria and of L. pulmonaria dried in low light to a PPFD of 800 μmol m−2 s−1. Damage to RCs was most severe in desiccated shade-grown L. pulmonaria which had, after prolonged predarkening, been dried in darkness. The observed sensitivity even of light-grown desiccated L. pulmonaria to illumination was not seen in other experiments, which used the method of stress application as in Fig. 6. This is perhaps the result of using a mercury lamp for illumination in the experiments of Table 3. The mercury lamp had more emission in the UV than the halogen lamp which was used in all other experiments.

Absorption changes at 800 and 950 nm and reversible loss of fluorescence in desiccated shade-grown thalli

Very strong illumination increased absorption at 800 and 950 nm reversibly both in desiccated sun-exposed and in shade-grown L. pulmonaria (see Fig. 7b,c for desiccated shade-grown L. pulmonaria). Fluorescence remained unchanged under strong illumination in desiccated sun-exposed L. pulmonaria, but some reversible quenching of chlorophyll fluorescence was observed in shade-grown thalli (Fig. 7a). The quantum yield of these reactions was very low. Radicals of chlorophyll are known to absorb at 800 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 reactions revealed fast and slower phases (not shown). The kinetics of the fast light-on and light-off phases were similar or 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 reaction and quencher formation. Whether the fast part of the 950 nm reaction is also related to quenching is less clear because of considerable noise of the 950 nm signal.

Figure 7.

 Reversible loss of chlorophyll fluorescence (a) of desiccated shade-grown Lobaria pulmonaria and reversible increase in 800 and 950 nm absorption (b, c) under illumination with a photosynthetic photon flux density (PPFD) of 11 000 μmol m−2 s−1. Arrows indicate onset (down arrow) and termination (up arrow) of illumination. In (a), loss of fluorescence F (%) is based on the fluorescence intensity of a desiccated thallus.


Loss of fluorescence during desiccation as indicator of the activation of thermal energy dissipation in L. pulmonaria

The need for protection of PSII RCs against excess light derives from the chemical reactivity of a few chlorophyll molecules within the RCs. Charge separation in the RCs initiates the use of light for photosynthesis, but leads to oxidative damage if excess light is not thermally dissipated (Barber & Andersson, 1992; Aro et al., 1993). Survival under strong light depends on the careful control of rogue reactions, such as the formation of chlorophyll triplet states and the ensuing transfer of excitation energy to oxygen which results in the formation of singlet oxygen. Efficiency of control is expressed by the extent of fluorescence quenching because chlorophyll fluorescence, energy conservation and energy dissipation are competitive processes. Extensive loss of fluorescence and of energy conservation during desiccation and in the dry state is therefore evidence of the activation of thermal energy dissipation.

However, other factors also contribute to the fluorescence decline upon desiccation. The cortex above the algal 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, consequently, fluorescence yield (Butler, 1962). There is also little doubt that shading of the algal layer below the cortex by melanic pigment relieves light stress on the algal photobionts of sun-grown L. pulmonaria (Gauslaa & Solhaug, 2001, 2004; Solhaug et al., 2003; McEvoy et al., 2007). Nevertheless, only a little fluorescence was lost during desiccation after a thallus had been treated with glutaraldehyde, an inhibitor of desiccation-induced fluorescence quenching (Fig. 2). Therefore, we conclude that increased attenuance in the cortex only contributes to the fluorescence loss in desiccated L. pulmonaria to a minor extent. Most of this effect is the result of fluorescence quenching by activation of thermal energy dissipation. Similar conclusions, based on fluorescence lifetime measurements, were drawn by Veerman et al. (2007) for the fluorescence decrease during desiccation of Parmelia sulcata.

Molecular basis of desiccation-induced energy dissipation

Previous investigations (Heber et al., 2007; Heber, 2008) have led to the conclusion that desiccation-induced fluorescence quenching results from desiccation-induced conformational changes of a chlorophyll protein. This conclusion was based on different observations: fast drying in darkness led to less fluorescence quenching than slow drying; fast drying decreased charge separation in PSII RCs less than slow drying; heating (intended to inactivate chlorophyll proteins by unfolding) increased fluorescence of rapidly dried desiccation-tolerant photoautotrophs less than that of slowly dried photoautotrophs; and glutaraldehyde inhibited desiccation-induced fluorescence quenching.

The present work with L. pulmonaria confirms and extends these observations. Strong desiccation-induced fluorescence quenching was also observed in L. pulmonaria. Its extent differed between shade- and sun-grown thalli (Fig. 1). Loss of fluorescence during desiccation was reversed when the desiccated thalli were heated (Fig. 3). Also here, different responses were observed for shade- and sun-grown thalli. Glutaraldehyde inhibited desiccation-induced quenching (Fig. 2).

In all lichens investigated so far, desiccation induced strong fluorescence quenching. This occurs not only in chlorolichens, which possess a chlorophyll b containing light harvesting complex, but also in the cyanolichen Peltigera neckeri, which does not (Heber et al., 2007). It therefore appears that the chlorophyll protein complex which changes its conformation upon drying is located within or close to the core of PSII, not in the antenna system. In cyanobacteria, c. 30 chlorophyll molecules reside in the core of PSII. They are in, or are close to, excitation equilibrium with the RCs of PSII (Mimuro & Kikuchi, 2003).

In lichens and desiccation-tolerant mosses, preferential quenching of the main emission of PSII fluorescence at c. 685 nm during desiccation is accompanied by far-red emission 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-induced fluorescence quenching. Strong support for this came from recent measurements of fluorescence life times (Veerman et al., 2007). The excitation life time of the far-red emitter was dramatically shortened by desiccation of the lichen P. sulcata. De-excitation of the bulk pool of excited chlorophyll was eight times faster after desiccation than in the hydrated state (Veerman et al., 2007). This drains excitation energy from the RCs, protecting them against photoinactivation.

Photoreactions were readily observable in desiccated shade-grown thalli as reversible photo-induced absorption changes at 800 and 950 nm. These changes were accompanied by kinetically closely related quenching of chlorophyll fluorescence (Fig. 7). Oxidation of chlorophylls is known to result in increased absorption at 800 nm. Reactive chlorophylls reside in the RCs of PSI and PSII. The RC of PSII contains six chlorophylls and two beta-carotenes. Light-dependent and reversible oxidation of a carotene has been observed in desiccated leaf fractions, which, in contrast to lichens, are accessible to transmission spectrophotometric analysis in the visible range (Shuvalov & Heber, 2003). For L. pulmonaria, Fig. 7(c) shows light-dependent absorption changes at 950 nm where carotenoid cations absorb (Holt et al., 2005). When oxidized, beta-carotene takes an electron from a neighbouring chlorophyll, ChlZD2. This produces a quencher (Faller et al., 2006). The similarity of the fast responses of fluorescence and of the 800 nm absorption change shown in Fig. 7(a,b) suggests formation of a quencher in the RC of PSII which could contribute to photoprotection by permitting energy dissipation within the RC. Significantly, the fast phase of the light reactions seen in photosensitive, desiccated, shade-grown L. pulmonaria as fast changes in 800 and 950 nm absorption was small or absent in the more phototolerant sun-grown L. pulmonaria.

The effect of light on desiccation-induced quenching

Low light, when present during desiccation, increased fluorescence quenching, heat-induced reversal of fluorescence quenching and phototolerance (Figs 1 and 3, Table 3). This was true both for the more photosensitive shade-grown and the more phototolerant sun-grown L. pulmonaria. The effect of the presence of light during drying has also been observed with other chlorolichens such as C. rangiformis and P. sulcata (Heber et al., 2007; Heber, 2008). For L. pulmonaria, it was recently reported by Stepigova et al. (2008).

How can the effect of light on desiccation-induced fluorescence quenching be explained? In hydrated higher plants, light controls the thermal dissipation of excess light energy that cannot be used for photosynthesis (Niyogi, 1999; Holt et al., 2004; Ruban et al., 2007). Necessary for activation of energy dissipation are the presence of the xanthophyll zeaxanthin (Demmig-Adams, 1990; Björkman & Demmig-Adams, 1994; Ahn et al., 2008) and the light-dependent protonation of a thylakoid protein, the PsbS-protein (Li et al., 2004; Takizawa et al., 2007). However, the protonophore nigericin which dissipates the ΔpH across the thylakoid membrane and inhibits the synthesis of zeaxanthin in the light did not inhibit desiccation-induced quenching in L. pulmonaria. The experiment of Fig. 5 shows that inhibition of zeaxanthin synthesis and of protonation of the PsbS protein by nigericin had little or no effect on the extent of quenching of fluorescence during desiccation of sun-grown L. pulmonaria.

Apparently, light is capable of interacting with the conformational changes of a chlorophyll protein which are induced by desiccation. Interaction could involve increased binding of zeaxanthin or other carotenoids. Table 2 shows that zeaxanthin was present in the thalli even after prolonged predarkening and desiccation of the thalli in the dark.

Sensitivity of desiccated L. pulmonaria to strong light

Gauslaa & Solhaug (1999, 2004, 2001) have reported light-induced damage to desiccated thalli of L. pulmonaria. Their observations are confirmed by the experiments shown in Fig. 6 and in Table 3, where partial loss of functionality of RCs after prolonged illumination of desiccated thalli, with light not even reaching full sunlight, is documented. More damage was inflicted to shade-grown than to sun-grown. L. pulmonaria. Also, thalli that had been dried in darkness were more damaged than those dried in the light, confirming results obtained by Stepigova et al. (2008).

Fluorescence was less quenched after desiccation of L. pulmonaria in darkness than after desiccation in the presence of light. Only a little light was required for increased fluorescence quenching during desiccation. Also, shade-grown thalli displayed less desiccation-induced quenching than sun-grown thalli. A negative correlation between light damage and fluorescence quenching is in line with the hypothesis that the latter is indeed photoprotective. One may argue that photodamage to L. pulmonaria is not entirely absent in desiccated thalli, pointing out that protection by desiccation does not exclude damage. However, other lichens, such as P. sulcata or Hypogymnia physodes, are more resistant to photoinhibition in the desiccated state than L. pulmonaria. Considering the sensitivity to light while water is still present (as shown by the almost total loss of PSII charge separation after slow desiccation in a PPFD of 300 μmol m−2 s−1 and ensuing illumination in the dry state (Fig. 6c)), the protection indicated by strong fluorescence quenching in the dry state is still dramatic, also in L. pulmonaria.

Adjustment of desiccation-induced energy dissipation to the irradiance

On desiccation, fluorescence was more quenched in sun-grown than in shade-grown L. pulmonaria (Table 1). It appears that photoprotection is regulated in L. pulmonaria both in the long term by growth and exposure conditions and in the much shorter term by the presence or absence of light during desiccation. Prolonged exposure to strong light during growth leads to slow melanin formation by the mycobiont as a sunscreen (Gauslaa & Solhaug, 2001) but also permits increased fluorescence quenching during desiccation (Fig. 1, Table 1). The molecular basis for increased fluorescence quenching in the sun-grown thalli is still unclear, but a main factor could be increased synthesis of desiccation-responsive chlorophyll protein. In the shorter term, availability of light during desiccation increases fluorescence quenching and, thereby, phototolerance (Table 3), probably by increased dissipative interaction between pigments within the desiccation-responsive protein. The interplay of these factors permits considerable flexibility in the adaptation to changing light stress conditions in the different seasons of a year as well as acclimation to habitats with different light climates (Gauslaa & Solhaug, 2000; Gauslaa et al., 2005).


UH wishes to acknowledge long-standing cooperation with Akademik V. A. Shuvalov, Russian Academy of Sciences, Pushchino-na-Oke, which helped to shape his views on the relationship between energy conservation and energy dissipation in photosynthesis. Professor R. Hedrich provided laboratory space and facilities at the University of Würzburg. We thank Dr. U. Schreiber, Dr. C. Klughammer and U. Schliwa for technical help. We are grateful to reviewers of a first submission of our work for their criticism. Mareike Jezek is thanked for skilful and patient help with the preparation of the figures.