f.PlantPhysiol. Vol. 134.pp. 261-268{1989}

Inhibition of Energy-Transfer to Photosystem II in Lichens by Dehydration: Different Properties of Reversibility with Green and Blue-green Phycobionts

W. BILGER*, S. RIMKE, U. SCHREIBER, and O. L. LANGE

Institut flir Botanik und Pharmazeutische Biologie der Universitat Wlirzburg, Mittlerer Dallenbergweg 64, D-8700 Wlirzburg, FRG * Present address: Carnegie Institution of Washington, Department of Plant Biology, 290 Panama Street, Stanford,

CA 94305

Received October 9, 1988 . Accepted December 12, 1988

Summary

Dry lichens with green algal phycobionts are able to recover net photosynthesis through rehydration with water vapor, whereas lichens with blue-green algae lack this ability (Lange, Kilian and Ziegler, Oeco­logia 71, 104 -110, 1986). By measurements of 77 K fluorescence emission and excitation spectra, it is in­vestigated whether this basic difference in rehydration properties of green and blue-green lichens is due to the different organization of the antenna pigments.

Emission spectra obtained from a lichen with a blue-green alga (Peltigera rufescens) and from a free-liv­ing blue-green alga (Nostoc d. commune) were essentially identical after normalization at 710 nm and re­vealed the following major features: Equilibration of a dry organism with air of 99 % r.h. resulted in a pronounced increase of fluorescence emission at 650 nm when excited at 580 nm (i.e. at maximal ab­sorbance for the phycobilin pigments). Additional spraying with liquid water led to a strong fluorescence increase around 690 nm. With excitation of chlorophyll at 430 nm no such changes could be observed with both procedures. Excitation spectra for emission at 695 nm revealed that the efficiency for fluo­rescence excitation at 565 nm compared to that at 435 nm was minimal after equilibration at 99 % r.h., whereas spraying with liquid water resulted in a strong enhancement. It is concluded that desiccation in­duces a functional detachment of the phycobilisomes (PBS) from photosystem II (PS II). Energy transfer from PBS to PS II is restored only when rehydration occurs with liquid water.

Similar experiments with the lichen Ramalina macifonnis, which contains a green phycobiont (Tre­bouxia spec.) yielded distinctly different responses. The desiccation induced lowering of fluorescence emission was almost totally reversed by equilibration with air of 93 % r.h. Excitation spectra of 695 nm emission showed a large reduction of the emission ratio with 480 nm excitation over that with 435 nm in­duced by desiccation. Rehydration by increasing air humidity resulted in a gradual increase of this ratio until it was almost maximal at about 99 % r.h. It is concluded that with green algal symbionts desiccation induces a functional interruption of energy transfer between the light harvesting chI alb pigment com­plex and PS II and that this can be largely restored by rehydration with humidified air, in contrast to the situation with blue-green algal symbionts.

Key words: Nostoc commune, Peltigera rufescens, Ramalina macifonnis, blue-green algae, chlorophyll fluo­rescence at 77 K, dehydration, energy transfer, lichens, photosynthesis, rehydration.

Abbreviations: LHCP = light harvesting pigment protein complex; PBS = phycobilisome(s); PS II (I) =

photosystem II (I); r.h. = relative humidity.

© 1989 by Gustav Fischer Verlag, Stuttgart

262 W. BILGER, S. RIMKE, U. SCHREIBER, and O. L. LANGE

Introduction

Lichens as poikilohydrous organisms are dependent in their life functions on the temporal availability of water. From former work (Butin 1954, Lange and Bertsch 1965, Bertsch 1966) it is known that lichens are able to take up the needed water even from the vapor phase. Recently, however, Lange et al. (1986) reported that only those lichens which contain green algae as phycobionts are able to restore photo­synthetic activity with water uptake from the vapor phase. In every lichen with blue-green algae examined so far, ir­respective of its thallus organization, even water potentials close to zero were insufficient to restore photosynthetic ca­pacity under such condition. However, rewetting of the li­chens by addition of liquid water resulted in an almost im­mediate increase of photosynthetic activity. There is evidence that this behavior is limiting to the performance of such lichens under field conditions as well (Lange and Ziegler 1986, Lange et al. 1988). So far, there has been no satisfactory explanation for the special requirement of liquid water for restoration of photosynthetic activity in blue­green lichens (see Lange et al. 1988).

It is apparent from the results of Lange et al. (1986, 1988) that the behavior of the lichens is determined by the given phycobiont and not by the fungal component. One special difference in the photosynthetic apparatus between green and blue-green algae is the organization of the light harvest­ing apparatus. Instead of a light harvesting chlorophyll pro­tein complex (LHCP), blue-green algae possess phycobilin containing particles, the phycobilisomes (PBS) (for recent re­views see Glazer 1984 and Zilinskas and Greenwald 1986). These are attached to the thylakoid membrane and reach into the lipid phase of the membrane, presumably via a small connection, the so-called anchor protein (Mimuro et al. 1986). Because of this structural feature energy transfer be­tween the PBS and PS II can be interrupted relatively easily, for example by cold treatment (Schreiber et al. 1979, Schreiber 1979), application of hydrostatic pressure (Schreiber and Vidaver 1973), or upon fragmentation of cells (Tel-Or and Malkin 1977).

Since the PBS contain the major part of light harvesting pigments of PS II (Tel-Or and Malkin 1977, Wang et al. 1977) energy transfer between both pigment protein complexes is essential for proper functioning of PS II and, hence, of electron transport activity. If functional PBS detachment were induced by desiccation, and if the detachment could not be reversed unless liquid water was applied, this could provide an explanation for the peculiar behavior of lichens with blue-green algae described previously by Lange et al. (1986).

Indeed, there is a report on desiccation induced PBS de­tachment in Collema flaccidium (Sigfridsson 1980). How­ever, in this work the rehydration behavior has not been in­vestigated. On the other hand, studies with red algae (Oquist and Fork 1982 a) led to the conclusion that in the desiccated state PBS transfer light energy preferentially to PS I. There­fore we reinvestigated energy transfer from PBS to PS II in lichens and blue-green algae, using fluorescence spectroscopy at 77 K and adopting the rehydration procedure used by Lange and Kilian (1985).

In this paper we present data which suggest that a func­tional detachment of PBS from PS II is indeed likely to be in­duced by desiccation in Peltigera rufescens, containing blue­green algae, as well as in Nostoc commune, and that this de­tachment is not reversed during water uptake from the gas phase. Only the addition of liquid water appears to restore a close contact of PBS and PS II. Interruption of energy trans­fer also was observed in dry thalli of the green algae contain­ing lichen, Ramalina maci/ormis. However, in this organism the loss of energy transfer between ChI band PS II is readily reversed by placing the lichen in humid air.

Material and Methods

Plant material

The experiments were performed with thalli of the lichen Petti· gera rufescens (Weiss) Humb. containing a blue-green Nostoc alga as a phycobiont. The specimens were collected in a «Mainfriinkischen Trockenrasen» near Markt Triefenstein (Lkr. Main-Spessart, West Germany). The free living blue-green alga Nostoc cf commune Vaucher was collected at the Botanical Garden in Wiirzburg. The lichen Ramalina macifonnis (Del.) Bory, with a green algae (Tre· bouxia spec.) phycobiont, was collected in the Negev Desert, Israel, and transported in the air dry state to Wiirzburg where it was stored in a deep freezer ( - 20°C) until use.

Treatment 0/ the lichens

Freshly collected lichens and algae (Peltigera r., Nostoc c.) or ma­terial taken from the deep freeze (Ramalina m.) was cleaned care­fully, rewetted and stored in the wet state for four days in a growth chamber. The temperature was controlled at 10°C and illumination provided by lamps (Osram HQL, 110/lmolm -2 S-1 photon fluence rate (400-700 nm), photoperiod 12: 12 hours). Following the pre­culture period the lichens were dried in an exsiccator over silica gel in complete darkness for three to four days, normally reaching a water content of about 5 to 10 % of the dry weight (<<dry» condi­tions). The lichens were subsequently rehydrated in gas exchange cuvettes as described Lange and Redon (1983). The humidity of the air was controlled by a cold trap and measured with dew point mir­rors (Walz, Effeltrich, FRG). The cuvette was thermostatized at 15°C by placing it in a waterbath (MGW, Lauda).

The relative humidity in the cuvettes was raised stepwise, be­ginning with 80 % and with further daily increases of 3 to 5 %. 24 h of exposure to a given r.h. was generally sufficient for stabilization of the water content as determined by weighing the samples. During this time the lichens were in darkness. After equilibration with air of 99 % r.h., the samples were sprayed with deionized water «<wet» conditions).

Measurement 0/ fluorescence spectra at 77 K

Fluorescence spectra at 77 K were measured with a modified Aminco SPF 500 spectrofluorometer equipped with fiberoptics and a dewar cuvette with a plane bottom window.

In order to avoid reabsorption artifacts, a «diluted powder» was prepared from the lichens, following the method outlined by Weis (1985). A fresh sample was removed with a corkborer (0 5 mm) and ground in a mortar under liquid nitrogen for at least 5 min. Subse­quently 1.8 g ice were added and grinding was continued until a ho­mogeneous sample was obtained. A small amount (40 mm3) of the resulting white powder was spread out by a cooled brass piston to give an approximately 1 mm layer on top of the dewar window,

interfaced from outside with the fiberoptics. The brass piston was then covered by liquid nitrogen.

Emission spectra were recorded with the excitation wavelength set at 430, 480 or 580 nm (bandpass 8 nm). To prevent artifacts caused by scattered excitation light the excitation beam was passed through a blue-green filter (BG 39, Schott, Mainz) and the photo­multiplier was protected by a red plastic filter (Roscolene No. 821, Edmunds Scientific). The bandwidth at the emission monochro-

Peltigera rufescens

Ekcitotion at 58Q nm Excitation at 430 om

dry dry

wet

660 700 740 780 660 700 740 780

• Wavelength. nm

Fig. 1: Fluorescence emission spectra at 77K of Peltigera ruleseens at different hydration states. The fluorescence was excited with a beam (bandpass 8 nm) centered at 580 nm or 430 nm. The dry lichen was desiccated over silica gel and had a relative water content of about 10 %. Rehydration of the lichen was induced by exposing a dry sample to humid air (99 % r.h.) (second row) and finally by spraying with liquid water (third row). The water content increased to 120 % of the dry weight in the sample at 99 % r.h. and to 680 % of the dry weight in the sprayed sample. The spectra are the mean of three re­plicas from different thallus pieces and are normalized at 710 nm. For further details see text.

Peltigera

Energy transfer in lichens 263

mator was 4 nm. Excitation spectra were recorded with a bandwidth of the excitation beam of 4 nm and in case of the measurements with blue-green algae, corrected for the effect of the BG 39 filter on the intensity of the excitation beam.

Operation of the spectrofluorometer, as well as spectra recording and analysis, was controlled by a Hewlett Packard HP 85 computer, connected to the spectrofluorometer via a programmable digital voltmeter (Keithley 192) and a lab-built interface. The emission spectra were corrected for the spectral sensitivity of the photomul­tiplier.

Results and Interpretation

Lichens with blue-green algae

Fig. 1 shows changes in the 77 K emission spectra of the blue-green lichen P. rufescens caused by rehydration of a de­siccated sample with preferential excitation of phycobilin (580 nm) or chlorophyll a (430 nm). Each spectrum was measured from an equal surface area of the lichen, but since the algal content was not constant and the density of the samples varied with moisture content, the absolute fluo­rescence yields cannot be compared. Before normalization, there was a general tendency of decreased yield with lower water content, the samples kept at 99 % r.h. displaying the lowest values. The presented spectra are normalized at 710 nm. This wavelength was chosen because the fluo­rescence yield at this wavelength was almost unvaried when the samples were excited at 430 nm. Although it is true, that the choice of normalization wavelength of the spectra may influence the resulting pattern shown in the difference spectra, the relative yield at different wavelengths is com­paratively insensitive against such manipulation. Also, it may be pointed out that normalization on the basis of the area of the spectra yielded almost identical results.

Phenomenologically, the spectra presented in Fig. 1 reveal two major features: First, the spectral changes observed upon rehydration were more pronounced with 580 nm ex­citation, i.e. when the phycobilins were preferentially ex­cited. Second, with 580 nm excitation, rehydration resulted

rufescens

Excitation at 580 nm Excitatian at 430 nm

~ III C Q)

99 % r.h. - dry 99 % r.h. - dry :::0 <1>

~ C <

<1> 0

..... c ~

-, <1> III

-, (')

Fig. 2: Difference spectra based on the fluo­rescence emission spectra of Peltigera rules· eens from Fig. 1. The first row shows the spectral changes obtained by the transition from dry conditions to storage in humid air, the second row shows the same for spraying a lichen kept at 99 % r.h. with liquid water. Fluorescence excitation at 580 nm or 430 nm, as indicated. For other conditions, see Fig. 1.

Q) u c Q) U III Q) .... o ::J ....

wet - 99 % r.h. <1> ::J wet - 99% r.h. (')

<1>

2-<1>

0 ::J III

.:< -,

660 700 740 780 660 700 740 760

Wavelength, nm

264 W. BILGER, S. R1MKE, U. SCHREIBER, and O. L. LANGE

in a strong stimulation of 690 nm fluorescence only when li­quid water was applied.

The 580 nm spectrum of the rewetted sample depicted in Fig. 1 was very similar to spectra usually obtained with fresh, wet samples and shows two minor peaks at 648 and 667 nm which can be related to emission from the phyco­bilin pigments, C-phycocyanin and allophycocyanin, respec­tively (Gantt 1981). Accordingly these peaks were missing when chlorophyll was preferentially excited (430 nm excita­tion light). PS II emission (around 690 nm; Rijgersberg and Amesz 1980) and PS I emission (at 720 and around 740 to 750nmj for a review see Fork and Mohanty 1986) were ob­served with both excitation wavelengths. In the case of pref­erential excitation of PS II by the phycobilin pigments with 580nm excitation (Wang et al. 1977, Zilinskas and Green­wald 1986), the relative amplitude of PS II emission exceeded that of PS I.

For a more detailed examination of the changes occurring with increasing water content, difference spectra were calcu­lated (Fig. 2). With excitation at 580 nm the difference be­tween the wet sample and the one kept at 99 % r.h. exhibited a dominant peak at 689 nm and two minima at 658 and 672 nm. Stimulation of 689 nm emission in the wetted sample clearly suggests restoration of energy transfer from the phycobilins to chlorophyll a. The fact that this was much less pronounced upon equilibration with water vapor favors the hypothesis that the liquid phase is required for reattachment of phycobilisomes, which became functionally disconnected from the thylakoid membrane by desiccation. The 658 nm change was presumably due to a decrease in phy­cobilin fluorescence, because it was not present with chloro­phyll excitation in contrast to the band around 672 nm. A band observed only with 580 nm excitation was induced around 655 nm, when the lichen was rehydrated from the dry state in humid air (99 % - dry). Changes in the long wave­length region were preferentially found with 430 nm excita­tion and presumably were originating from PS I pigments.

Results obtained with a free living blue green alga, Nostoc commune, were quite similar to those with P. rufescens (Fig.

Nostoc commune

Excitation at 580 nm Excitation at 430 nm

>-.... VI

99"10 r.h. - dry 99"10 r.h. - dry C (II

~ (II u c (II u -1 VI (II L.. 0 :::l wet - 99"10 r.h. wet - 99"10 r.h. ;;::

(II > 0 0 Qj 0::

-1 660 700 740 780 660 700

Wavelength, nm

3). Again the most prominent change, around 690 with 580 nm excitation, occurred with the addition of liquid water to the lichen. This is particularly evident from the difference spectra shown in Fig.4.

Another means of probing energy transfer between the phycobilin pigments and PS II are measurements of excita­tion spectra for PS II fluorescence. The 77 K spectra shown in Fig. 5 were recorded at an emission wavelength of 695 nm with samples of P. rufescens at different states of hydration. The spectra displayed two peaks, one for the direct excita­tion of chlorophyll a with a maximum at 435 nm, and a sec­ond one for excitation of phycoerythrin at 565 nm (Gra­bowski and Gantt 1978). The height of the 435 nm peak tended to be rather constant for all degrees of hydration

Nostoc commune

E)(citation at 58Q nm Excitation at 430 nm

d" d"

660 700 TLO 780 660 700 TLO 780

Wavelength. nm

Fig. 3: Fluorescence emission spectra at 77K of Nostoc commune at different hydration states. Fluorescence excitation at 580 nm or 430 nm, as indicated. Further conditions as in Fig. 1.

0

-1

o

740 780

::0 C1>

[ <' C1>

~ c 0 ..., C1> VI n C1> :::l n C1>

Fig. 4: Difference spectra of the fluorescence emission spectra of Nostoc commune shown in Fig. 3. Fluorescence excitation at 580 nm or 430nm, as indicated. For other condi­tions, see Figs. 1 and 2.

whereas the 565 nm peak showed large variations. This peak first was somewhat decreased when a desiccated sample was equilibrated with water vapor, and it was strongly increased again (by about a factor of 4) upon wetting the sample with liquid water. This behavior is further characterized for the complete set of measurements in Fig. 6, which shows ratios of fluorescence yield excited by 565 and 435 nm light at in­creasing water content. The results of an equivalent experi­ment with N. commune are depicted (open bars). The most significant change was the stimulation of PS II excitation via phycoerythrin absorption when the samples were rewetted, suggesting, as with the fluorescence emission data presented above, that the liquid water phase is essential for reestablish­ment of a functional contact between the PBS and the thyla­koid membrane. The relatively high PS II excitation with 565 ~m light in the dry sample will be dealt with in the Dis­cussion.

Lichens with green algae

Lange and coworkers have shown (1985, 1986) that lichens containing green algae are very different from those contain­ing bluegreens with respect to their capacity for recovery of photosynthesis upon dehydration. Green algal lichens are able to regain most of their capacity for photosynthetic COruptake in an atmosphere of high humidity without moistening through liquid water. As will be shown below, such capability is also reflected through fluorescence meas­urements.

Peltigera rufescens

Emission at 695 nm

2 dry

~ 'iii c 4>

0 C

4> u C 4> u III 4>

0 .... 0 :::> -4> > :a 2 4i c::

600

Excitation wavelength, nm

Fig. 5: Fluorescence excitation spectra at 77 K of Peltigera rufescens at different hydration states. The emission wavelength was set at 695 nm (bandpass 4 nm). The samples were those used in the experi­ment of Fig. 1. The spectra are the mean of three replicas from dif­ferent thallus pieces and were corrected for the change in excitation beam intensity with wavelength.

Energy transfer in lichens 265

6

~ II> P. rufescens ..., ~ e- 7 [J N. commune ...... IS) to IS)

e- 6 Ill' "0 4i '>' 5

OJ u c 4 OJ U III OJ

0 3 :::> -4i l-

e .2 "0 c::

dry 90% 93% 99% wet

Fig. 6: Ratio of emission yields with excitation by 565 or 440 nm light for samples in different hydration states. The lichens or algae were either desiccated, equilibrated with humid air of different r.h., or rewetted with liquid water, as indicated. The open bars represent measurements with N commune, the closed ones show the results obtained with Peltigera rufescens. The fluorescence yield values were derived from spectra similar to those shown in Fig. 5. The emission was measured at 695 nm. Where standard deviations are shown, the values represent the means of 3 to 11 replicas.

Fluorescence spectra of dry thalli of Ramalina maci/ormis reveal a very small absolute fluorescence yield of about 15 % of samples fully hydrated by liquid water. However, when kept at a relative humidity of 90 %, resulting in a water con­tent of 30 % of the dry weight, the fluorescence yield recov­ered to values close to those obtained with the wet control (data not shown). The spectra shown in Fig. 7 A, for a sequence of rehydration steps, were normalized at 750 nm. In Fig. 7B the corresponding difference spectra are dis­played. A major change in the emission spectrum occurred with rehydration of the dry sample at 93 % relative humidity and a water content of 36 % (as measured in a parallel sample). Notably, and in contrast to the behavior of the blue-green lichen, there were only minor spectral changes when the samples rehydrated in humid air were wetted. The relative proportions of the fluorescence peaks at 686 and 698 nm (PS II) and the shoulder around 715 nm, which is characteristic for PS I emission in green algae (Govindjee and Satoh 1986), did not vary significantly between 93 % relative humidity and full wetting of the lichen. The first water uptake resulted in a decrease maximal at 676 nm and an ac­companying increase peaking at 700 nm. With further water uptake the maximum is shifted to a wavelength of 686 nm. This feature points to a slight change in energy distribution between PS II and PS I, which has been found to occur upon drying in other lichen species (Sigfridsson and Oquist 1980, Jensen and Feige 1987). Rewetting with liquid water pro­duced no specific changes, except for a further decrease around 670 nm.

266 W. BILGER, S. RIMKE, U. SCHREIBER, and O. L. LANGE

Ramalina maciformls

dcy A B

93-'. r.h - dry

wet ~ 99 -I. r.h

---------.------."' .. ~~---- 0

wet -, 660 700 740

Wavelength, nm

660 700 740

Wavelength, nm

Fig. 7: Fluorescence emission spectra at 77 K from Ramalina maci­/ormis at different hydration states. Excitation at 480 nm (bandpass 8 nm). The lichen thalli were desiccated, equilibrated with air of the indicated relative humidity, or sprayed with liquid water. The spectra represent the means of three replicas from different thallus pieces and are normalized at 750 nm. A: Original emission spectra. B: Difference spectra of the various emission spectra shown in A.

Similar spectra have been obtained upon rehydration of dry samples of the free-living aerophilous green alga Pleuro­coccus spec. (data not shown). This, again, points to the fact, that the rehydration behavior of the lichen is determined by the phycobiont.

Excitation spectra with R. maci/ormis for the fluorescence emission at 695 nm (Fig. 8) show that in the dry state PS II was mainly excited around 438 nm, which is characteristic for chlorophyll a absorption. In the wet state the band at 480 nm due to chlorophyll b absorption predominated. Chlorophyll b is the main pigment of the light harvesting complex (LHC) of green algae and higher plants. The ratio of the excitation efficiency at 480 to that at 438 nm increased gradually with increasing degree of hydration (Fig. 9). Hence, it is suggested that in the desiccated state energy transfer from the light harvesting chI a/ b complex to chI a is particularly affected and gradually restored with increasing rehydration. It may be pointed out that a very similar recov­ery behavior of R. maci/ormis upon rehydration was found for overall COz-fixation (Lange, O. L., unpublished results, compare Lange and Bertsch 1965).

Discussion and Conclusion

The presented fluorescence data confirm previous results obtained by gas exchange measurements that lichens with

blue-green algae have a special liquid water requirement for restoration of photosynthetic activity after drought stress. In addition, the information from fluorescence emission and excitation spectra can give new insight into the possible mechanistic basis of the peculiar rehydration behavior of blue-green lichens. The data lead to the conclusion that in blue-green as well as in green lichens, desiccation causes a

~ III C G>

C

G> u C G> u III G> o .2 G> > § G>

a:::

Ramalina maciformis

1.5 Emission at 695 nm

1.0

0.5

400 450 500

Excitation wavelength, nm

Fig. 8: Fluorescence excitation spectra of representative samples of Ramalina maci/ormis in the dry and rewetted state. Emission wave­length, 695 nm (bandpass 4 nm). The spectra are normalized at 438 nm. For further conditions see legend to Fig. 7 and text.

Ramalina maciformis CI)

'" ~ e- 1.6 ..... 0 <D ~

1.4 e-Ill'

" 1.2 ] :>.

G> 1.0 -u c: G>

0.8 u III G>

0 0.6 ~

~ 0.4

.... 0 0.2 .2 C; 0 a::: dry 90% 93% 99% wet

Fig. 9: Ratio of emission yields with excitation by 480 nm or 435 nm light in thalli of Ramalina maciformis at different rehydration states. The lichens were either desiccated, equilibrated with humid air, or rewetted with liquid water, as indicated. Where standard deviations are shown, the fluorescence yield ratios represent mean values of 5 to 10 replicas. For measuring conditions, see Figs. 7 and 8.

functional detachment of the major light harvesting pigment complexes from photosystem II; however, only in green algal lichens restoration of energy transfer takes place by equilibration with humid air. In the following paragraphs the results leading to this conclusion are discussed in more detail.

The spectral data presented in Figs. 1-6 clearly indicate that in the blue-green organisms a major change occurred upon the transition from an almost water saturated at­mosphere to liquid water, resulting in the restoration of en­ergy transfer between the phycobilin pigments and chi a. In particular the excitation efficiency of PS II fluorescence with 565 nm light, which is absorbed mainly by phycoerythrin (Gantt 1981), increased strongly as compared to direct excita­tion of chlorophyll at 435 nm (Fig. 6). With restoration of energy transfer from the PBS to PS II one expects a decrease of the emission of allophycocyanin at 660 and 680 nm and an increase in PS II emission at 685 and 695 nm. These changes were indeed observed (Figs. 2 A and 4 A). An alternative ex­planation for an increase in PS II fluorescence with rewetting could be a selective quenching of PS II emission in the desic­cated state. However, this explanation can be ruled out since the changes around 690 nm were very small when chloro­phyll was excited (Figs. 2 Band 4 B). With free living N. com· mune the spectral changes between 650 and 700 nm were rather small when excitation was at 430 nm. For the algae in P. rufescens this was true for the 655 nm band, and to a lesser extent also for emission at 695 nm, whereas the pronounced minimum at 675 nm in the difference spectrum was present with excitation at 430 nm as well. On the basis of the pre­sented data it is difficult to offer an explanation for the changes at this wavelength. However, it appears likely that some change of chlorophyll protein complexes is involved.

Surprisingly, in the totally dry state energy transfer from PBS to PS II was affected to a lesser extent than at 90 - 99 % r.h. as shown in Fig. 6. This finding was corroborated fur­ther by the occurrence of a pronounced peak at 650 nm in the difference of the emission spectra for the samples equi­librated at 99 % Lh. and the dry ones (Figs. 2 and 4). This suggests that, upon shrinkage of the organisms with severe desiccation, the PBS may somehow reestablish close contact with the membrane. In this context the finding of Oquist and Fork (1982 a) is important, that in Porphyra thalli, which were mechanically prevented from shrinking upon desicca­tion, energy transfer from phycoerythrin to PS I was low­ered compared to shrunken samples.

Sigfridsson (1980) reported a decreased energy transfer be­tween phycoerythrin and PS II when Collema jlaccidium was desiccated at 50% r.h. However, in his work no attention was given to the rehydration procedure. Our results are in agreement with Sigfridsson's finding that in the dry state the PBS has no contact to either photosystem. A similar result has been obtained by Oquist and Fork (1982) with the red alga Porphyra per/orata. However, in their findings the de­crease in energy transfer from phycoerythrin to PS II was ac­companied by an increase of transfer to PS I. The latter was attributed to changes in the membrane structure due to water loss and could be prevented by stretching the algae during desiccation.

In the lichen with green algae the functional connection between PS II and the light harvesting antenna pigments was

Energy transfer in lichens 267

also lowered through desiccation. This was clearly shown by the excitation spectra (Figs. 8, 9) for PS II emission at 695 nm. Similar results were obtained by Sigfridsson (1980) for Cladonia impexa, whereas Oquist and Fork ( 1982 b) could not find pronounced changes in the relative efficiency of 433 nm and 478 nm light for excitation of fluorescence at 685 nm in the liverwort Porella navicularis and the isolated lichen alga Trebouxia pyriformis. Possibly the occurrence of this process is dependent on the species and its respective habitat. The similarity between the recovery behavior of en­ergy transfer from LHCP to PS II and of COrfixation in R. maciformis (Lange and Bertsch 1965) suggests that energy capture by the antenna pigments is limiting for the overall process. Alternatively, light harvesting efficiency could have been adjusted to match the energy demand for the dark reac­tions, avoiding photoinhibition.

As already shown by gas exchange measurements, the de­siccation-induced changes are reversible by uptake of water vapor in the green organisms, but not in the blue-green ones. Our data indicate that in this respect the free-living algae and those in the lichens behave very similarly. This finding fur­ther corroborates the earlier assumption that the ability to recover photosynthesis by water vapor uptake is determined by the phycobiont and is independent of the characteristics of the mycobiont (Lange et al. 1986, 1988).

The question arises as to what extent the changes in PBS at­tachment upon desiccation and rehydration can be the cause for the observed changes in photosynthetic activity. As the chlorophyll content of PS II is relatively low in blue-green algae (Tel-Or and Malkin 1977, Mimuro and Fujita 1977), PBS are the main antenna for PS II excitation. If effective en­ergy transfer between these pigment protein complexes is in­hibited, one should expect PS II excitation to be severely de­creased and consequently electron transport to be suppressed. Hence, a close contact between PBS and PS II may be considered a necessary requirement for efficient photosynthesis. The excitation spectra presented in Fig. 5 do not allow a precise quantification of the desiccation induced lowering of the absorption cross section of PS II, but indicate that PBS detachment is not complete. However, in the state reached by equilibration with air of 99 % Lh., PBS detach­ment certainly is sufficiently severe to cause a clear-cut lim­itation of electron transport capacity. Therefore, PBS attach­ment should be considered as a possible site for inhibition by desiccation stress in blue-green algae in addition to the sites already discussed for other organisms (see Kaiser 1987). Fur­ther studies are needed to clarify the relative contribution of PBS detachment to the overall limitation of photosynthesis. The general question as to why the PBS are functionally re­attached only following imbibition with liquid water must remain open at this point. The presented results point to the fact that the addition of liquid water is accompanied by modifications in membrane structure. This might be due either to the amount of water which is necessary to induce the structural changes or to the status of liquid water which allows a solution to be reformed after desiccation (see Lange et al. 1988). The nature of these processes has still to be cla­rified in order to obtain a better understanding of the pro­cesses which control photosynthesis in blue-green algae under conditions of water stress.

268 W. BILGER, S. RIMKE, U. SCHREmER, and O. L. LANGE

Acknowledgements

The authors thank Christian Neubauer for help with the meas­urements and Dr. William Adams for correction of the language. This work was supported by the Deutsche Forschungsgemein­schaft.

References

BERTSCH, A.: Uber den COrGaswechsel einiger Flechten nach Was­serdampfaufnahme. Planta 68, 157 -166 {1966}.

BUTIN, H.: Physiologisch-okologische Untersuchungen tiber den Wasserhaushalt und die Photosynthese bei Flechten. Biolo­gisches Zentralblatt 73, 459-502 (1954).

FORK, D. C. and P. MOHANTY: Fluorescence and other character­istics of blue-green algae (Cyanobacteria), red algae, and crypto­monads. In: GOVINDJEE, J. AMESZ, and D. C. FORK (eds.): Light Emission by Plants and Bacteria, 451- 496. Academic Press, Or­lando {1986}.

GANTT, E.: Phycobilisomes. Ann. Rev. Plant Physiol. 32, 327 - 347 {1981}.

GLAZER, A. N.: Phycobilisome. A macromolecular complex op­timized for light energy transfer. Biochim. Biophys. Acta 768, 29-51 (1984).

GOVINDJEE and K. SATOH: Fluorescence of green and brown algae. In: GOVINDJEE, J. AMESZ, and D. C. FORK (eds.): Light Emission by Plants and Bacteria, 497 - 537. Academic Press, Orlando (1986).

GRABOWSKI, J. and E. GANTT: Photophysical properties of phycobi­liproteins from phycobilisomes: Fluorescence lifetimes, quantum yields, and polarization spectra. Photochem. Photo­bioI. 28, 39-45 {1977}.

JENSEN, M. and G. B. FEIGE: The effect of desiccation and light on the 77 K fluorescence properties of the lichen Peltigera aphthosa. Bibl. Lichenol. 25, 325-330 (1986).

KAISER, W. M.: Non-stomatal, primary dehydration effects on photosynthesis: Possible mechanisms for reversible and irre­versible damage. Current Topics in Plant Biochemistry and Physiology 6, 119-133 (1987).

LANGE, O. L. and A. BERTSCH: Photosynthese der Wtistenflechte Ra­malina maciformis nach Wasserdampfaufnahme aus dem Luft­raum. Naturwissenschaften 52,215-216 (1965).

LANGE, O. L., T. G. A. GREEN, and H. ZIEGLER: Water status related photosynthesis and carbon isotope discrimination in species of the lichen genus Pseudocyphellaria with green or blue-green photobionts and in photobiosymbiodemes. Oecologia 75, 494-501 {1988}.

LANGE, O. L. and E. KILIAN: Reaktivierung der Photosynthese trockener Flechten durch Wasserdampfaufnahme aus dem Luft­raum: Artspezifisch unterschiedliches Verhalten. Flora 176, 7 - 23 (1985).

LANGE, O. L., E. KILIAN, and H. ZIEGLER: Water vapor uptake and photosynthesis of lichens: Performance differences in species with green and blue-green algae as phycobionts. Oecologia 71, 104-110 (1986).

LANGE, O. L. and J. REDON: Epiphytische Flechten im Bereich einer chilenischen «Nebeloase» (Fray Jorge). II: Okophysiologische Charakterisierung von COrGaswechsel und Wasserhaushalt. Flora 174, 245-284 (1983).

LANGE, O. L. and H. ZIEGLER: Different limiting processes of photo­synthesis in lichens. In: MARCELLE, R., H. CUJSTERS, and M. VAN POUCKE (eds.): Biological Control of Photosynthesis, 147 -16l. Maninus Nijhoff Publishers, Dordrecht (1986).

MIMURO, M. and Y. FUJITA: Estimation of chlorophyll a distribution in the photosynthetic pigment systems I and II of the blue-green alga Anabaena variabilis. Biochim. Biophys. Acta 459, 376-389 {1977}.

MIMURO, M., C. LIPSCHULTZ, and E. GANTT: Energy flow in the phy­cobilisome core of Nostoc sp. (MAC): Two independent terminal pigments. Biochim. Biophys. Acta 852, 126-132 (1986).

OQUIST, G. and D. C. FORK: Effects of desiccation on the excitation energy distribution from phycoerythrin to the two photo­systems in the red alga Porphyra perforata. Physiol. Plant. 56, 56-62 (1982 a).

- - Effects of desiccation on the 77 K fluorescence properties of the liverwort Porella navicularis and the isolated lichen green alga Trebouxia pyriformis. Physiol. Plant. 56, 63 -68 (1982 b).

RIJGERSBERG, C. P. andJ. AMESZ: Fluorescence and energy transfer in phycobiliprotein-containing algae at low temperature. Biochim. Biophys. Acta 593, 261-271 (1980).

SCHREmER, U.: Cold-induced uncoupling of energy transfer between phycobilins and chlorophyll in Anacystis nidulans. Antagonistic effects of monovalent and divalent cations, and of high and low pH. FEBS Letters 107, 4-9 (1979).

SCHREIBER, U., C. P. RIJGERSBERG, and J. AMEsz: Temperature-de­pendent reversible changes in phycobilisome-thylakoid mem­brane attachment in Anacystis nidulans. FEBS Letters 104, 327 -331 (1979).

SCHREmER, U. and W. VIDAVER: Photosynthetic energy transfer re­versibly inhibited by hydrostatic pressure. Photochem. Photo­bioI. 18, 205-208 (1973).

SIGFRIDSSON, B.: Some effects of humidity on the light reactions of photosynthesis in the lichens Cladonia impexa and Collema /lac­cidium. Physiol. Plant. 49, 320-326 (1980).

SIGFRIDSSON, B. and G. OQUIST: Preferential distribution of excita­tion energy into photosystem I of desiccated samples of the lichen Cladonia impexa and the isolated lichen-alga Trebouxia py­riformis. Physiol. Plant. 49, 329-335 (1980).

TEL-OR, E. and S. MALKIN: The photochemical and fluorescence properties of whole cells, spheroplasts, and spheroplast particles from the blue-green alga Phormidium luridum. Biochim. Bio­phys. Acta 459, 157 -174 (1977).

WANG, R. T., C. L. R. STEVENS, and J. MYERS: Action spectra for photo reactions I and II of photosynthesis in the blue-green alga Anacystis nidulans. Photochem. Photo bioI. 25, 103-108 (1976).

WEIS, E.: Chlorophyll fluorescence at 77 K in intact leaves: Charac­terization of a technique to eliminate artifacts related to self-ab­sorption. Photosynth. Res. 6, 73 - 86 (1985).

ZIUNSKAS, B. and L. S. GREENWALD: Mini Review: Phycobilisome structure and function. Photosynth. Res. 10,7-35 (1986).