Photosynthates stimulate the UV-B induced fungal anthraquinone synthesis in the foliose lichen Xanthoria parietina

Plant, Cell and Environment

(2004)

27

, 167–176

© 2004 Blackwell Publishing Ltd

167

Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 20032004

272167176Original Article

Photosynthate stimulation of lichen compound synthesisK. A. Solhaug & Y. Gauslaa

Correspondence: Yngvar Gauslaa. E-mail: [email protected]

Photosynthates stimulate the UV-B induced fungal anthraquinone synthesis in the foliose lichen

Xanthoria parietina

K. A. SOLHAUG & Y. GAUSLAA

Department of Ecology and Natural Resource Management, Agricultural University of Norway, P.O.Box 5003, NO-1432 Ås, Norway

ABSTRACT

Synthesis of the cortical anthraquinone pigment parietin (=physcion) was studied in acetone-rinsed, parietin-free

Xan-thoria parietina

thalli. UV-B induced the synthesis, whichincreased linearly with UV-B (log-transformed) to thehighest applied UV-B level (1.8 W m----

2

). At natural UV-Blevels (0.75 W m----

2

), parietin resynthesis occurred at a con-stant pace (106 mg m----

2

d----

1

) during a 14-d period at220

mmmm

mol m----

2

s----

1

PAR. Under these conditions, 56% of thenatural parietin content prior to extraction was resumed,accounting for 10% of total net carbon gain. In the pres-ence of UV-B, the remaining results were consistent withthe hypothesis assuming that photosynthates regulate thepace at which parietin is synthesized by the mycobiont.Resynthesis was rapid when photosynthesis was activatedby light, or when certain carbohydrates were added exoge-nously. Additions of ribitol, the carbohydrate deliveredfrom the photobiont, increased the parietin resynthesis sub-stantially. Mannitol, the main fungal polyol, was signifi-cantly less effective. Furthermore, parietin resynthesis in

X.parietina

was depressed at high and low hydration whennet photosynthesis is depressed. Therefore, the photobiontregulates the parietin resynthesis pace in its mycobiontpartner by the delivery of photosynthates. In conclusion,both lichen bionts play important roles in the synthesis ofparietin, which probably acts as a PAR- rather than a UV-B-screen.

Key-words

: acetone rinsing; carbohydrates; lichen symbio-sis; photosynthesis; photosynthetically active radiation;physcion; secondary compounds; UV.

INTRODUCTION

The fungal partner in the symbiotic relationship of a lichenthallus often produces large amounts of secondary com-pounds, as reviewed by Fahselt (1994). The secondarychemistry of lichens is well studied (e.g. Huneck &Yoshimura 1996), and has long been acknowledged as auseful tool by lichen taxonomists. The ecological signifi-

cance of these secondary lichen compounds is less clear;several functions, such as irradiance screening, chemicaldefence against herbivores and parasitic fungi, have beenproposed (Lawrey 1986; Huneck 1999). There are few con-clusive experimental studies on the induction, productionand ecological significance of secondary compounds; andmany ecological studies are so far equivocal. For example,in studies of the irradiance screening function of lichencompounds, some found a positive relationship betweenirradiance and concentrations of UV-B absorbing second-ary compounds (Rundel 1969; Legaz

et al

. 1986; Bjerke,Lerfall, & Elvebakk 2002; Buffoni-Hall, Bornman, &Björn 2002), whereas others did not (Stephenson & Run-del 1979; Golojuch & Lawrey 1988; Swanson, Fahselt, &Smith 1996).

Lack of data, inconsistent data, and the many differentassumed ecological roles of lichen compounds (Elix 1996;Lawrey, Torzilli, & Chandhoke 1999; Lawrey 2000;Hyvärinen

et al

. 2000; Hyvärinen, Walter, & Koopmann2002) make it especially important to study the effect ofenvironmental factors on the induction and production ofsecondary compounds by experimental approaches. Con-trasting results in the literature might not only be causedby different roles and/or induction factors for differentcompounds, but also by different experimental approaches.Recent methodological innovations have facilitated exper-imental studies on the induction and production of second-ary lichen compounds in an intact lichen thallus. Secondarycompounds can be extracted from air-dry thalli of manyliving lichen species with acetone without affecting meta-bolic activities of the organisms (Solhaug & Gauslaa 1996;Lange

et al

. 1997; Solhaug & Gauslaa 2001). Acetone-extracted, compound-free lichen thalli (Fig. 1) offer uniqueopportunities to study in detail how various external factorsregulate the resynthesis of secondary compounds. Recently,Solhaug

et al

. (2003) used this method to show unambigu-ously that UV-B triggered the resynthesis of the brightlyorange anthraquinone pigment parietin (= physcion) in thelichen

Xanthoria parietina

(L) Th. Fr. (Fig. 1). Importantly,conclusive and consistent results were obtained both undernatural field conditions as well as in growth cabinets withartificial photosynthetically active radiation (PAR) andUV-B. Additional, ecological field studies have found astrong correlation between parietin content and site factors

168

K. A. Solhaug & Y. Gauslaa

© 2004 Blackwell Publishing Ltd,

Plant, Cell and Environment,

27,

167–176

reflecting the openness of the habitat, suggesting that solarradiation plays a crucial role (Hill & Woolhouse 1966;Gauslaa & Ustvedt 2003). However, a natural sun-shadegradient is complex; other environmental variables corre-late with the inherently tightly coupled PAR and UV-Birradiances. Therefore, we still need detailed experimentalstudies in order to document and understand how environ-mental factors influence the synthesis pace of secondarycompounds in intact thalli. Even for the well-studied pari-etin, the question whether it serves as a UV-B- or PAR-screening pigment, has not yet been finally answered(Gauslaa & Ustvedt 2003).

Lichens with high concentrations of secondary com-pounds necessarily invest a significant amount of carbon forthe synthesis of these substances. Therefore, the lichen pho-tobiont might be expected to play an important role forcompound synthesis as the main supplier of assimilates ina lichen thallus. Photobiont photosynthesis has alreadybeen suggested to regulate mycobiont respiration (Palm-qvist

et al

. 2002). Evidence for such a hypothesis is availablefor some lichen species, as reviewed by Fahselt (1994). Thephotobiont of

X. parietina

is the green alga

Trebouxia arbo-ricola

(Beck, Friedl & Rambold 1998). Studies of

Xanthoriacalcicola

, which is closely related to

X. parietina

(Scherrer

& Honegger 2003), show that its photobiont translocatesfixed carbon in the form of ribitol to the mycobiont (Rich-ardson & Smith 1968a, b). Ribitol is the exported carbohy-drate from common photobionts such as

Coccomyxa,Myrmecia

and

Trebouxia (

reviewed by Ahmadjian 1993),and can be a major leachate also in other lichens (Cooper& Carrol 1978). Subsequent to its uptake, the mycobiontquickly converts ribitol to the photobiont-inaccessible man-nitol via the pentose phosphate pathway (as reviewed byAhmadjian 1993; Palmqvist 2000). An isolated

X. parietina

mycobiont produces parietin on culturing media containingribitol (Honegger & Kutasi 1989). However, we do not yetknow how exogenously added carbohydrates affect theparietin resynthesis in the symbiotic phenotype of an ace-tone-rinsed parietin-free lichen thallus.

This study aimed to target stimulating factors for themycobiont’s parietin synthesis in the widespread folioselichen

X. parietina

. In this symbiotic lichen association,parietin is exclusively found in the peripheral part of theupper cortex (Honegger 1990). Our main hypothesis is thatthe photosynthetic assimilation of carbon in the photobiontregulates the pace at which parietin is synthesized by themycobiont, provided a required UV-B induction of themycobiont (Solhaug

et al

. 2003). According to this hypoth-

Figure 1.

The photo shows one air-dry

Xanthoria parietina

thallus on a sea cliff. The cortical orange pigment parietin has been extracted by acetone from the left piece, showing a grey colour that occa-sionally can be observed in shady natural habitats. The right thallus piece exhibits its natural and common parietin colour. The molecular structure, and the absor-bance spectrum of parietin redissolved in ethanol are shown in the graph. There is no absorbance between 500 and 700 nm (data not shown).

Photosynthate stimulation of lichen compound synthesis

169



27,

167–176

esis, reductions in net photosynthesis due to lack of PAR,to desiccation, or to the substantial suprasaturation depres-sion recorded in

Xanthoria

(Lange & Green 1996), shouldstop or slow down the parietin synthesis. Furthermore, inthe absence of photosynthate production due to lack ofPAR, the hypothesis also implies that artificially addedassimilates in terms of carbohydrates should enhance theparietin synthesis. If photosynthates can be shown to stim-ulate parietin production, the last requirement (listed byCockell & Knowland 1999) for ascribing a PAR-protectionrole to parietin has been complied with. Finally, we also aimat establishing the dependency of parietin synthesis on theUV-B level, and to document the kinetics of parietin pro-duction in parietin-free thalli.

MATERIALS AND METHODS

Lichen material

Xanthoria parietina

thalli were collected from open sun-exposed sea cliffs at Strömstad, W Sweden (58

∞

52

¢

N,11

∞

7

¢

E) and Jeløy, Moss, SE Norway (59

∞

26

¢

N, 10

∞

36

¢

E)(see Table 1). Each collection consisted of several differentthalli from many different rock surfaces. The numerousthalli were air-dried at room temperature and stored max-imally a few weeks at

-

20

∞

C until the start of the experi-ments. A batch of thalli for one experiment was alwaystaken from one collection occasion only (Table 1), and eachbatch contained similar-looking, healthy pieces with similarcolour and amount of apothecia. Thalli were divided into

3–5 cm

2

pieces like those illustrated in Solhaug

et al

. (2003),and not more than three to four pieces were taken fromone single thallus, often less. Pieces from all sampled thalliin one batch were well mixed. Ten pieces were randomlyselected for each separate treatment.

Parietin extraction and measurement

Air-dry

X. parietina

-thalli were acetone-rinsed four timesin order to extract and quantify the amount of parietin(Solhaug & Gauslaa 1996). However, to obtain a nearlycomplete extraction of parietin, the standard extractiontime was extended from 5 to 10 min (Solhaug

et al

. 2003).Even with these prolonged extraction times, we found that3.8% of the original parietin content was left in test thalliby applying several additional repeated acetone extrac-tions. Therefore, 3.8% was subtracted from all measuredvalues after resynthesis to compensate for residues of pari-etin after the standard extraction procedure. The acetonedid not extract significant amounts of other compoundsthan parietin, as the computed parietin concentration (1–2% of dry mass) based on measured absorptance and theextinction coefficient of parietin (Solhaug & Gauslaa 1996)fully corresponded to measurements of extract weight sub-sequent to evaporation of acetone (data not shown). Thespectrum of a freshly made acetone extract of

X. parietina

(330–700 nm; see Solhaug & Gauslaa 1996), showed thatthe extract from desiccated thalli did not contain eventraces of chlorophylls. No immediate (Lange

et al

. 1997;Solhaug & Gauslaa 2001) and long-term adverse effects

Table 1.

A short description of all experiments with specified combinations of treatments

ExperimentCollection dateand location Wetting regime

UV-B,(W m

-

2

)PAR,(

m

mol m

-

2

s

-

1

)Duration,(d)

Partietin contentat start (g m

-

2

)

UV-B-level April 6, 2002 Spraying withdistilled water(hydration 20 h daily)

0.015 220 7 2.02

±

0.05Strömstad 0.15 220 7 (

n

= 60)0.230.350.801.80

220220220220

7777

Time course April 6, 2002 Spraying withdistilled water(hydration 20 h daily)

0.75 220 1 2.65

±

0.09Strömstad 0.75 220 2 (

n

= 60)0.750.750.750.75

220220220220

48

1421

Hydration April 28, 2001 Spraying withdistilled water(hydration 10/24 h daily)

0 180 5 3.25

±

0.11

¥

UV-B Jeløya 0.75 180 5 (

n

= 40)

Carbohydrates March 16, 2002 Soaking in variouscarbohydrate solutions (5 g L

-

1

)0.75 0 7 2.18

±

0.06Strömstad (

n

= 60)PAR May 26, 2001 Soaking in ribitol solutions 0 0 7 3.30

±

0.07

¥

UV-B Strömstad (0, 2 or 5 g L

-

1

) 0 220 7 (

n

= 120)

¥

ribitol 0.750.75

0220

77

The last column shows the content of parietin in the various collected samples prior to acetone extraction, means

±

1 SE are given. Althoughthe different samples had different parietin content at the time of collection, subsamples for each treatment (

n

= 10) within one experiment,did not differ significantly in parietin content (

ANOVA

).

170

K. A. Solhaug & Y. Gauslaa



27,

167–176

(Solhaug & Gauslaa 1996; Solhaug

et al

. 2003) of acetoneextraction have been found for this symbiotic lichen asso-ciation, probably because lichen compounds are located ascrystals outside the hyphae, and because acetone appar-ently does not enter desiccated cells.

In all experiments, the parietin was expressed as percent-age parietin after resynthesis compared with the parietincontent of thalli collected in the field. The parietin contentin thalli collected from the field varied significantly betweencollection events (2.02–3.30 g m

-

2

; Table 1). Parietin wasexpressed per unit thallus area, since this parameter is morefunctionally relevant than per weight, especially for thiscortical compound. The thallus area was measured in thehydrated state with a leaf area meter (LI3100; Licor Inc.,Lincoln, NE, USA). The natural parietin content prior toextraction did not significantly differ between the subsam-ples used for different treatments within any of the fiveexperiments described in Table 1 (

ANOVA

; data not shown).After extraction, thalli were left over night to let remain-

ing acetone evaporate. Afterwards, as a standard precondi-tioning to the various experiments (see below), thalli werehydrated and kept in the hydrated state under low incan-descent light (approximately 3

m

mol m

-

2

s

-

1

) for 48 h at18

∞

C.

Experimental conditions

All experiments were performed in a growth room at con-stant air temperature of 12

∞

C. Photosynthetically activeradiation was given as an 18 h photoperiod at 220 or180

m

mol m

-

2

s

-

1

(Table 1) from mercury metal halide lamps(Osram Powerstar

®

HQI-BT 400 W daylight; Osram AS,Norway). The PAR level is somewhat below the PAR sat-uration of net photosynthesis (unpublished data). Six hoursUV radiation from UV-B fluorescent tubes (TL 20 W/12RS;Esshå Elagentur AB, Värnamo, Sweden) was given in themiddle of the daily 18 h photoperiod, UV-B radiation wasmeasured with a UV-B sensor (model SKU 430; Skye Instru-ments Ltd, Llandrindod Wells, Powys, UK). Different UV-B levels (Table 1) were obtained by manipulating the num-ber of UV-B tubes and the distance from the tubes. Celluloseacetate film (Rachow, Hamburg, Germany; 0.10 mm, 50%cut-off at 295 nm) was used for all UV-B treatments toeliminate possible UV-C. Thalli not exposed to PAR wereshielded by black cloth. The 220

m

mol m

-

2

s

-

1

PAR regimegave a daily PAR dose of 14.3 mol m

-

2

d

-

1

, which is about50% of typical daily PAR dose in the field in June, and themost common UV-B irradiance applied (0.75 W m

-

2

) wasapproximately the same as typical daily field levels in June(16 kJ m

-

2

d

-

1

; see Solhaug

et al

. 2003 for field levels of PARand UV-B). Therefore, thalli subjected to this irradianceregime received twice as much UV-B relative to PAR in thegrowth cabinet than under natural field conditions.

Five thalli were placed upon three layers of wet filterpaper in each 10 cm Petri dish. The Petri dishes were placedupon five layers of wet filter paper in open top plastic boxes(30 cm

¥

20 cm

¥

5 cm) to reduce the evaporative demand.Boxes and Petri dishes were rotated every day to reduce

possible effects of minor spatial variations in irradiance.Screening filters (cellulose acetate film) were placed hori-zontally 1 cm above the upper edge of the boxes to allowfree air circulation.

Different hydration regimes were applied. Thalli in thehydration experiment (Table 1) were either kept continu-ously wet by placing the hydrated thalli on soaking wetfilter papers, or kept hydrated for a shorter period by plac-ing moistened thalli on dry filter papers. These thalliremained hydrated maximally for 10 h a day. All thalli inall experiments were wetted once daily immediately beforethe onset of UV-B exposure. Thalli in the remaining exper-iments described in Table 1 were moistened by eitherspraying by water or soaking for 2 min in carbohydratesolution, and remained moist and photosynthetically activemost of the time until wetting next day. The amount ofwater added to the filter papers daily had been adjusted toallow thalli to become dry shortly before wetting on thefollowing day, since the hydration experiment and somepreliminary experiments have shown that constant hydra-tion is less favourable for parietin production than onedaily desiccation event. To obtain comparable hydrationperiods under different PAR-depending evaporativedemands, more water was added to the PAR-exposed thallithan to thalli kept in darkness.

Chlorophyll

a

fluorescence

Chlorophyll fluorescence was measured after the precondi-tioning in the hydrated state under low incandescent light(approximately 3

m

mol m

-

2

s

-

1

) for 48 h at 18

∞

C with a por-table fluorometer [plant efficiency analyser (PEA); Hansat-ech, King’s Lynn, Norfolk, UK]. The

F

v

/

F

m

values werecalculated by the instrument from fluorescence inductioncurves of 5 s duration recorded at an irradiance of1500

m

mol m

-

2

s

-

1

from light-emitting diodes. Such mea-surements were performed prior and subsequent to theUV-B-level and the factorial PAR

¥

ribitol

¥

UV-B experi-ments in order to study whether applied irradiances signif-icantly affected the lichen photobiont. No significantvariation in maximum dark-adapted photosystem II effi-ciency existed between lichen treatments prior to exposureaccording to the

ANOVA

(data not shown).

RESULTS

UV-B level

Resynthesis of parietin in

X. parietina

thalli, exposed toUV-B for 1 week, increased linearly with the logarithmicvalue of the UV-B irradiance (

r

2

= 0.952,

P

< 0.001) up tothe highest applied UV-B irradiance (1.8 W m-2; Fig. 2a).Because of the logarithmic response along the whole testedUV-B range, no upper and lower UV-B threshold levels forthe parietin resynthesis were detected. At the highest UV-B exposure, which was twice the normal maximum levelsduring clear summer days at the latitude for lichen collec-tions, 42.4% of the original parietin content of2.02 ± 0.05 g m-2 (mean ± standard error; Table 1) was

Photosynthate stimulation of lichen compound synthesis 171

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 167–176

resynthesized within 7 d. This is equivalent to an averageresynthesis rate of 122 mg m-2d-1. The 6 h daily UV-B expo-sures were given to moist and continuously metabolicallyactive thalli, whereas high UV-B under natural field condi-tions is normally associated with rapid inactivation of pho-tosynthesis due to rapid drying. Therefore, the biologicallyrelevant UV-B dose at the highest UV-B irradiance(Fig. 2a) was higher than the ecologically realistic dose.Thalli exposed to normal UV-B irradiances under naturalboreal daylight conditions (0.15–0.80 W m-2), resynthesized26–39% of their original parietin content (Fig. 2a). Thalliexposed to traces of UV-B (0.015 W m-2) resynthesizedonly 6% parietin.

No long-lasting adverse effects of the applied UV-Bexposures on the photobiont could be detected in spite ofextraction of the UV-B absorbing parietin shield. The max-imum dark-adapted photosystem II efficiency was at similarlevel before (0.720) and after (0.740) UV-B exposures.Measured as percentage of initial Fv/Fm values, this param-eter did not differ between the tested UV-B exposures(ANOVA) after 2 d recovery subsequent to the exposureexperiment (Fig. 2b). Already after 40 min recovery, Fv/Fm

values had recovered substantially (mean including all UV-B treatments: 0.637).

Time course of parietin synthesis

Resynthesis of parietin occurred at a constant pace of106 mg m-2 d-1 during the first 14 d period, and parietin

resynthesis could be clearly detected already after 1 d(Fig. 3). An extended regression line nearly reached theorigin (0.035% at time zero). The very close correlationallowed comparisons of parietin resynthesis betweengrowth cabinet experiments using different cultivation peri-ods. At day 14, the parietin content had reached 56% of thestart level at 2.65 ± 0.09 g m-2 (mean ± standard error;Table 1).

The next and final measurement was taken after 21 d, butno further increase occurred during the third week(48.3 ± 3.8%, not included in Fig. 3). The reason for theweak decline after the longest cultivation period is notknown. Possibly, resynthesis might have reached an equi-librium at the current environmental conditions. On theother hand, the conditions in the growth cabinet mightpossibly have negative long-term effects. For example,repeated spraying with distilled water might have leachedthe mineral nutrients from the cultivated thalli.

Hydration regime

Hardly any parietin resynthesis occurred in the absence ofUV-B, regardless of hydration regime (Fig. 4). UV-B wasclearly required for the induction of parietin synthesis(Table 2). For those treatments receiving UV-B, there wasa highly significant effect of hydration (thehydration ¥ UV–B interaction in Table 2). More parietinwas resynthesized with continuous hydration compared tothe parietin resynthesis in thalli that experienced extensivedesiccation during the daylight period (Fig. 4). However,substantially less resynthesis occurred in continuously wetthalli (Fig. 4) in comparison with thalli that experienced aminor desiccation period, as those displayed in Fig. 3.Therefore, parietin resynthesis apparently declined at bothlow and very high hydration levels, reaching a maximalpace at some intermediate hydration regimes.

Figure 2. Parietin resynthesis in percentage of original concen-tration before acetone extraction (a) and maximum dark-adapted PSII efficiency as percentage of start values (b) in Xanthoria pari-etina thalli after 7 d cultivation at 220 mmol m-2 s-1 PAR (18 h pho-toperiod) with various levels of UV-B irradiation (6 h per day). The error bars show ±1 SE (n = 10). Initial Fv/Fm values were not significantly different between UV-B levels (ANOVA; overall mean Fv/Fm value was 0.720).

Figure 3. Time course of parietin resynthesis (percentage of orig-inal concentration before acetone extraction) in Xanthoria pariet-ina thalli cultivated at 220 mmol m-2 s-1 PAR (18 h photoperiod) and 0.75 W m-2 UV-B (6 h per day). The error bars show ±1 SE (n = 10) when larger than symbol.

172 K. A. Solhaug & Y. Gauslaa


Carbohydrate source

Parietin resynthesis depended on the type of carbohydratethat was exogenously added by daily submersion of thalliin carbohydrate solutions during cultivation with UV-B, butno PAR, for 7 d (Fig. 5; P < 0.0001; ANOVA). Very littleparietin resynthesis occurred in the control thalli beinghydrated with pure water. Insignificantly higher resynthesisof parietin occurred with sorbitol solution compared to thatof pure water. Both glucose and mannitol yielded signifi-cantly higher parietin contents than those of sorbitol andpure water, whereas the largest resynthesis took place inthalli receiving either ribitol or sucrose (Fig. 5). However,even the best carbohydrate sources (ribitol or sucrose)resulted in a lower parietin resynthesis rate (45 mg m-2 d-1)than the 220 mmol m-2 s-1 PAR treatment used in mostother experiments (Figs 2 & 3).

UV-B ¥¥¥¥ PAR ¥¥¥¥ ribitol interaction

UV-B was the most influential factor for the resynthesis ofparietin (P < 0.001), followed by PAR (P < 0.001), and thenribitol (P = 0.018) according to the three-way ANOVA

(Table 3) applied on the factorial experiment shown inFig. 6. The highest parietin resynthesis occurred inthe + PAR + UV-B treatment with the highest ribitol addi-tion (23.3%), the lowest in the absence of all three factors(Fig. 6). The best treatment resulted in an average resyn-thesis rate of 110 mg m-2 d-1. Ribitol had the cleareststimulating effect in the absence of PAR, especially whenUV-B was given (Fig. 6). The presence of UV-B strength-ened the stimulating effect of both PAR and ribitol.

The experiment recorded in Fig. 6 was a replicate, butsomewhat modified experiment of another 5 d experimentat lower PAR (90 mmol m-2 s-1) with only two ribitol treat-

Figure 4. Parietin resynthesis (percentage of original concentra-tion before acetone extraction) in Xanthoria parietina thalli culti-vated for 5 d at two hydration regimes and two UV-B regimes at 180 mmol m-2 s-1 PAR (18 h photoperiod). Black columns: about 10 h daily hydration, open columns: continuous hydration (24 h). UV-B regimes: 0.75 W m-2 6 h per day, or no UV-B). The error bars show 1 SE (n = 10), and means with the same letter were not significantly different at P < 0.05 by the Student–Newman–Keul method. ANOVA-result: see Table 2.

Figure 5. Parietin resynthesis (percentage of original concentra-tion before acetone extraction) after 7 d in Xanthoria parietina thalli cultivated at UV-B (0.75 W m-2, 6 h per day) in darkness. All thalli were once every day soaked in solutions with 5 g L-1 of various carbohydrates. Means were significantly different (P < 0.0001, ANOVA). The error bars show 1 SE (n = 10), and means with the same letter were not significantly different at P < 0.05 by the Student–Newman–Keul method.

Table 2. Two-way ANOVA of parietin resynthesis in Xanthoria parietina thalli cultivated for 5 d at two hydration regimes and two UV-B regimes at 180 mmol m-2 s-1 PAR (18 h photoperiod)

Source d.f. F-ratio P

Hydration regime 1 9.689 0.004UV-B 1 81.177 0.000Hydration regime ¥ UV-B 1 16.134 0.000Error 36

Parietin resynthesis was log-transformed, log(1 + parietinpercentage), to fit the distribution requirements of the ANOVA.R2 = 0.748. Mean values are shown in Fig. 4.

Table 3. Three-way ANOVA of parietin resynthesis in Xanthoria parietina thalli cultivated for 7 d in a factorial experiment with two UV-B levels (0 or 0.75 W m-2 6 h per day), two PAR levels (0 or 220 mmol m-2 s-1 PAR, 18 h photoperiod) and three ribitol levels (0, 2 or 5 g L-1)

Source d.f. F-ratio P

UV-B 1 163.026 0.000PAR 1 24.479 0.000Ribitol 2 4.197 0.018UV-B ¥ PAR 1 6.339 0.013UV-B ¥ ribitol 2 3.444 0.035PAR ¥ ribitol 2 0.797 0.453UV-B ¥ PAR ¥ ribitol 2 0.310 0.734Error 108

R2 = 0.662. Mean values are shown in Fig. 6. No transformations ofvariables were needed to satisfy the ANOVA requirements.



ments (0 and 2 g L-1; data not shown), and lichen materialfrom the same collection as in the hydration experiment(Table 1). All other conditions were similar. The resynthesiswas lower in this slightly shorter experiment with less light(10.5% of start level for the treatment with UV-B, PAR,and ribitol; equivalent to 68 mg m-2 d-1). Nevertheless, thisexperiment gave the same effects and trends as thosereported in the main experiment (Fig. 6, Table 1).

The 220 mmol m-2 s-1 PAR treatment in the main experi-ment (Fig. 6) increased the level of long-term photoinhibi-tion, as the Fv/Fm-values were reduced from 0.758 ± 0.004(–PAR; n = 60) to 0.679 ± 0.005 (+PAR; n = 60) even whenmeasured after 48 h recovery at low light subsequent to thetreatment. PAR was the only clearly significant factor(P < 0.001) for Fv/Fm according to a three-way ANOVA (datanot shown). Therefore, the two Fv/Fm means mentionedabove included all UV-B and ribitol treatments without andwith PAR, respectively. However, the parietin resynthesisincreased with PAR in spite of the recorded PAR-inducedlevel of photo-inhibition.

DISCUSSION

Our results show that acetone-rinsed, compound-deficientX. parietina thalli are well suited for studying regulatingfactors for the synthesis of parietin. This method allowstesting of old hypotheses dealing with induction and syn-thesis of secondary compounds in lichens. Induction studieson lichens with a natural content of various compounds canbe impeded by reported UV-B dependent degradation ofsome secondary lichen compounds (BeGora & Fahselt2001a). It is difficult to interpret experiments when achanged content of compounds might depend on anunknown balance between compound synthesis, degrada-

tion, and dilution due to thallus growth, as might have beenthe case in some earlier induction studies on lichens (e.g.BeGora & Fahselt 2001b). In comparison with studies onisolated and cultured mycobionts, the mycobiont in anintact, but compound-deficient thallus operates in a physi-ologically more relevant context than isolated and lessstructured mycobionts cultured aposymbiotically. There-fore, an experiment using compound-deficient lichen thalliis a useful supplement to synthesis studies on isolatedmycobionts.

Solhaug et al. (2003) showed that hardly any parietinresynthesis takes place in X. parietina in the absence of UV-B. Apparently, there is no threshold level of the inductivepotential of the UV-B irradiance within ecologically rele-vant doses (Fig. 2a), including also future UV regimes atpredicted atmospheric ozone depletions (McKenzie et al.2003). UV-B strongly influences the parietin resynthesis,although the logarithmic response (Fig. 2) emphasizes theimportance of fluctuations at low UV-B levels. The parietinsynthesis takes place in the mycobiont (e.g. Ahmadjian1993), and the mycobiont in the upper cortex of a lichenthallus is a highly efficient UV-B screen for underlyingphotobionts, even in the absence of cortical pigments(Gauslaa & Solhaug 2001). Therefore, the UV-B regulationof parietin resynthesis most probably also occurs in themycobiont. The strong UV-B regulation might easily leadto the conclusion that the main role of parietin is to protectthe lichen against excessive UV-B, according to the fulfil-ment of three out of four required criteria listed by Cockell& Knowland (1999). However, the lacking evidence of UV-B induced damage in a Xanthoria mycobiont even underconditions simulating the outer space environment (deVera et al. 2003) and the photobiont (Fig. 2b) supports ear-lier suggestions and/or conclusions that Xanthoria is a UV-B resistant lichen genus (Nybakken et al. in press).

More data supports a PAR- than an UV-B-protectivefunction. A natural parietin screen protects X. parietinaphotobiont against excessive PAR damage (Solhaug &Gauslaa 1996) by efficiently screening the 400–500 nm PARband width (see Fig. 1 and Gauslaa & Ustvedt 2003). Thesignificantly higher PAR susceptibility of the symbiotic X.parietina photobiont subsequent to acetone-extraction sug-gests a role also for the photobiont in the parietin resyn-thesis. Provided a given amount of UV-B irradiance,substantial data (Figs 4–6) are consistent with our mainhypothesis assuming that the net photosynthetic assimila-tion of carbon in the photobiont regulates the pace at whichparietin is synthesized by the mycobiont. The photobiontphotosynthesis has already been suggested to regulate themycobiont respiration (Palmqvist et al. 2002). Parietinresynthesis is higher when the photosynthesis is activatedby light than in darkness where respiration consumesstored photosynthates, and it increases under the influenceof exogenously added carbohydrates (Fig. 6). No parietinresynthesis occurs in desiccated thalli (Solhaug et al. 2003).Furthermore, the hydration dependence of net photosyn-thesis in X. parietina is similar to that of X. calcicola (Langeet al. (1996) and Lecanora muralis (Lange 2002) according

Figure 6. Parietin resynthesis (percentage of original concentra-tion before acetone extraction) after 7 d in Xanthoria parietina thalli in a factorial experiment with two UV-B levels (0 or 0.75 W m-2 6 h per day), two PAR levels (0 or 220 mmol m-2 s-1 PAR, 18 h photoperiod) and three ribitol levels (0, 2 or 5 g L-1). The thalli were once every day soaked in water or ribitol solutions. The error bars show 1 SE (n = 10). ANOVA-result: see Table 3.



to O.L. Lange (personal comm.). These lichens exhibit astrong suprasaturation depression at high hydration levels.In fact, X. parietina was among the most severely affectedlichens during an extraordinarily wet period when a severedie back occurred (Gauslaa 2002). Many lichens requirealternate periods of drying and wetting for maintaininghigh rates of photosynthesis and large pools of polyols (Far-rar 1976b). An intermediate level of hydration was used inmost of our experiments, which probably gave a clearlyhigher net production of photosynthates, and thereby alsoa significantly higher parietin resynthesis than the high andlow hydration regimes applied in the hydration experiment(Fig. 4). Unfortunately, we were not able to study the PAR-dependence of parietin resynthesis in detail, since a changein PAR inevitably implies a significant change in hydrationregime. Nevertheless, the increase in PAR from 90 mmolm-2 s-1 in the first factorial experiment (see text above) to220 mmol m-2 s-1 in the final experiment (Fig. 6) approxi-mately doubled the parietin resynthesis. Such data suggeststhat the PAR response is more linear than the UV-Bresponse. Therefore, and because of a strong correlation innature between PAR and UV-B, the regulatory role of PARmight well be substantial under natural conditions. In thisway, the final unresolved induction criterion of Cockell &Knowland (1999) for a screening role of PAR might beconsidered fulfilled; the other criteria dealing with absorp-tion, screening efficiency, and protection from damage werealready met by Solhaug & Gauslaa (1996), Gauslaa &Ustvedt (2003) and Solhaug et al. (2003). Lacking evidenceof the protection criterion suggests that UV-B screening isnot the primary function of parietin.

The strong dependency of parietin resynthesis uponphotosynthate production suggests that parietin mighthave a function as a sink of excess carbon, which was pro-posed as a role of secondary compounds by Mosbach(1973). A rough estimate based on net photosynthesismeasurements 1–2 mmol CO2 m-2 s-1 (unpublished results)and a parietin synthesis rate of 106 mg m-2 d-1 (calculatedby means of the data from Fig. 3) shows that as much as10% of net carbon gain may have been deposited as pari-etin. Therefore, parietin might not only protect againstPAR by its screening function (Solhaug & Gauslaa 1996);it might also possibly reduce oxidative stress at high lightby being a sink to which excess photosynthates can beconverted (see Öquist & Huner 2003) without spendinglimited sources of N.

The rate of carbohydrate uptake and transfer from pho-tobiont to mycobiont must be fast, since parietin resynthe-sis is already measurable within 1 d. Parietin resynthesissubsequent to acetone extraction starts shortly after expo-sure to UV-B, and occurs at a constant pace during the first14 d under experimental conditions (Fig. 3). We do notknow the natural carbohydrate pool in the pre-conditionedthalli prior to experimental conditions, but it possiblybecame depleted during the 48 h pre-conditioning at con-stant hydration, low PAR, and relatively high temperature(see Farrar 1976a). However, the fast resynthesis rate iscompatible with existing reports of relatively rapid and sub-

stantial conversion of fixed carbon in Xanthoria calcicolainto mannitol during a 24-h period (Richardson & Smith1968a) or less (Lines et al. 1989). Considering the complex-ity of the metabolic processes involved: import of ribitol,conversion of ribitol to mannitol, synthesis of parietin,transfer and deposition of parietin as crystals outsidehyphae of the conglutinated upper cortex; the speed atwhich parietin accumulates in measurable amounts (Fig. 3),is surprisingly high. Honegger (1986, 1991) suggests that atleast over short distances, mycobiont-derived secondarymetabolites are translocated in a yet unknown form (pos-sibly glycosides), and crystallize within and on the hydro-phobic wall surface layer of hyphae. The operational timescale of the parietin sunscreen regulation is compatible withpossible seasonal acclimations in nature. Because of thestrong steering by UV-B (Fig. 2a) and PAR (Fig. 6), theparietin sunscreen might be strengthened most quicklyunder optimal conditions for net photosynthesis. So far, theseasonal acclimation of the parietin screen has apparentlynot been measured, but the reported parietin resynthesisrate might even allow adjustments at a time scale of a clearweather period lasting for some days provided there aresome hydration events due to morning dew or short rainyevents.

The released carbohydrate from the X. parietina photo-biont, ribitol, is one of the two most efficient of the testedcarbohydrates for the parietin resynthesis (Fig. 5) whenthese are added exogenously. Arabitol, a major fungalpolyol in many green-algal lichens (Dahlman et al. 2003),might possibly also have been efficient if applied. Sorbitoland glucose, the carbohydrates released from other greenalgal and cyanobacterial photobionts, respectively(Ahmadjian 1993), were significantly less effective. Suchcarbohydrate specificity could be one reason for the well-documented mycobiont dependency on one particularphotobiont genus. However, whereas aposymbiotically cul-tured X. parietina mycobiont needs ribitol foranthraquinone production, aposymbiotically culturedmycobionts of other Xanthoria species might produceanthraquinones on a wider range of carbon sources(Honegger & Kutasi 1989). Therefore, results in Fig. 5 arenot necessarily valid for other species. Various responses ofexogenously added carbohydrates for the synthesis of otherlichen compounds in other taxonomic groups, as reviewedby Fahselt (1994), suggest that different species and com-pounds respond differently. However, acetone-extractedand compound-deficient thalli of other lichen species havenot yet been studied.

In conclusion, compound-deficient acetone-extractedthalli of X. parietina are well suited for the study of induc-tive factors for parietin resynthesis. UV-B irradiance is thestrongest induction factor for parietin resynthesis in themycobiont of an intact X. parietina thallus. However, thephotobiont regulates the parietin resynthesis pace in itsmycobiont partner through its delivery of photosynthates.Therefore, both lichen bionts play important roles in thesynthesis of parietin, which probably acts as a PAR screenrather than a UV-B screen.



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Documents

Photosynthates stimulate the UV-B induced fungal anthraquinone synthesis in the foliose lichen Xanthoria parietina