Biochemical Systematics and Ecology, Vol. 14, No. 3, pp. 283-286, 1986. 0305-1978/86 $3.00+0.00 Printed in Great Britain. Pergamon Journals Ltd.

Factors Affecting Two Different Usnic Acid Dehydrogenases in the Lichen Evernia prunastri

CARLOS VICENTE, AZUCENA GONZALEZ and MARIA ESTRELLA LEGAZ Department of Plant Physiology, The Lichen Team, Faculty of Biology, Complutense University, 28040 Madrid, Spain

Key Word Index--Evernia prunastti; Usneaceae; lichen; usnic acids; dehydrogenases; light; desiccation; enzyme induction. Absttact--Evernia prunastri accumulates D-usnic acid which can be remobilized by two different enzymes. One of them, DL-usnic acid dehydrogenase, behaves as a constitutive protein which is removed from the cell membrane to the cytoplasm when the lichen suffers a progressive desiccation. D-Usnic acid dehydrogenase is synthesized as a response to increased concentrations of its substrate, D-usnic acid.

Introduction Thalli of Evernia prunastri are able to remobilize D-usnic acid (Fig. 1), which is accumulated in their medulla, when the lichen is under critical

conditions of dryness and nutrient availability [1]. Light accelerates the catabolism of this phenol. This process seems to be started by an usnic acid dehydrogenase which uses both D- and L-

~H3 CO

ICH3 H3C C / ~ "CO CH 3 OH H OH

CO

O ~ l j O H D-Usnic acid f NAD(P)÷ / ,~3C " L ' ~ H dehydrogenase

(~H 3 / ' OH ~ (r H3 CH3 ~" NAD' P'H co ~ " ~ co ~o , , 2 .o o. .o o

3 " Y X ~ 3 " Y H3C" Y " 3 3 " Y 1"13 c/ Y " 3 H C H C CO-CH H C CO-CH OH " ~ OH. OH OH " 0

H3C" " ~ CO-CH3 dehydrogenase

OH ~NAD* ~H3 !

CO H O ~ O H

H3C" ~r ~ H3C" ~ "CO CH 3 OH 0

FIG. I. SCHEME OF THE BIOSYNTHESIS OF USNIC ACIDS AND THE PROPOSED CATABOLIC REDUCTION BY USNIC ACID DEHYDROGI::NASES IN EVERNIA PRUNASTRI.

(Received 4 May 1985)

284 CARLOS VICENTE, AZUCENA GONZALEZ AND MARIA ESTRELLA LEGAZ

forms of this compound but only uses NADH as cofactor [2]. Moreover, enzyme activity is always very low when recently collected thalli are used as a source of the protein. A second usnic acid dehydrogenase has been described [Vicente and Gonzalez, submitted] although this enzyme only reduces D-usnic acid by using both NADH and NADPH as cofactors. However, the affinity of the enzyme for the first nucleotide is about 30 times higher than that found for the second one. This new enzyme is only produced when lichen thalli are supplied with exogenous D-usnic acid.

Both dehydrogenases are really used to re- mobilize D-usnic acid accumulated by E. prunastri thalli, although they catalyse their respective reverse reactions as all the dehydrogenases act. However, their biosynthetic role is dubious since, as described by Taguchi eta/. [3], oxidative reac- tions in the biosynthetical pathway of usnic acids only affect to methylphloroacetophenone units to form the carbon-carbon bond in the furanic ring (Fig. 1), whereas the remobilizing enzymes act on the constituted heterocyclic molecule.

This catabolism of the phenol, performed by the lichen itself, is very different to that carried out by soil microorganisms [4-6] which enzy- matically remove the acetyl groups at the 6 position from the phenol.

In this work, we attempt to study the condi- tions in which both usnic acid dehydrogenases are produced by E. prunastri thalli.

Results The time-course of DL-usnic acid dehydrogenase activity is shown in Fig. 2. Enzyme activity increases up to 5 days of maintenance of lichen thalli in dry conditions under white light and this enhancement of the activity is not inhibited by 100 ~M cycloheximide infiltrated inside the thalli. After 5 days, dehydrogenase activity rapidly decreases to practically zero at 14 days treat- ment. Red light apparently produces an inhibi- tory effect on the development of enzyme activity which, on the other hand, does not appear when the thalli are maintained under continuous darkness or floating on distilled water, in the light as well as in the dark.

To discern whether the effect of red light is a photomorphogenetic response or a subsidiary thermal action on lichen metabolism, dry thalli were floated on polyethylene glycol (PEG; aver-

14

_12

5 'to

E 8

__>-++

m Z+

?: ~ 2

o'

J l //

2 Z, 6 8 10 12 14 days

FIG. 2. TIME-COURSE OF eLUSNIC ACID DEHYDROGENASE ACTIVITY IN EVERNIA PRUNASTRI. Lichen thalli were maintained under white light (o) or red light (11) in dry conditions at 26 ~, When dry thallus samples were maintained in the dark, or floated on distilled water in the dark as well as in the light, enzyme activity has not been detected (V). Cycto- heximide infiltrated inside the thalli (A) maintained under white light does not significantly change enzyme activity. Values are the mean of three replicates. Vertical bars give standard error where larger than the symbols.

age molecular weight 600) at different concen- trations under red light at three times lower value of photon flux rate (5 p, mol m 2 s-l) than that used in drying out experiments. Results are shown in Fig. 3. The progressive desiccation produced by increasing concentrations of PEG enhances dehydrogenase activity although this effect is reversed by treatments longer than one day when high concentrations of PEG are used, or three days for the lowest value of osmotic pressure. The insert A in Fig. 3 shows that DL- usnic acid dehydrogenase is easily extracted when thalli are treated with increasing concen- trations of Triton X-100 before buffer extraction. This indicates that this dehydrogenase behaves as a particulate enzyme which can be dissolved after membrane solubilization.

Changes in D-usnic acid dehydrogenase are summarized in another way in Fig. 4. Enzyme activity appears as a response to the presence of D-usnic acid in the culture medium, whereas it is not developed when thalli are floated on bicar- bonate alone. The highest value of enzyme activity is reached by thalli maintained for 12 h under white light, although it can also be detected when incubations are carried out in the

USNIC ACID DEHYDROGENASES IN EVERNIA PRUNASTRI 285

e ",:-°0 o.s ,0 ,.s 2 . 0 / - - / ~per cent Triton X-:.~//

/ i \ : ; - :

l ~ v I • I • I v----'-T'V

0( 200 400 600 800 I PEG) rnosmol

FIG. 3. TIME-COURSE OF DL-USNIC ACID DEHYDROGENASE ACTIVITY OF THALLI FLOATED ON PEG SOLUTIONS. Incubations were carried out under low intensity of red light for 1 (e), 2 (A), 3 (11) and 4 (T) days at 26 ° . The insert shows the recovery of enzyme activity from thallus samples maintained in dry conditions under low intensity of red light and pre-treated with variable concentrations of Triton X-100 before extraction. Values are the mean of three replicates. Vertical bars give standard error where larger than the symbols.

40 o i

~ 30

8 ~ _ 0 2 /, 6 8 10 12 14

hours FIG. 4. TIME-COURSE OF DL-USNIC ACID DEHYDROGENASE ACTIVITY Thallus samples were floated on 0.35 mM 0-usnic acid under white light (o) or in the dark (e). When incubations are carried out on 2% bicar- bonate or 0.35 mM D-usnic acid+100 IdVI cycloheximide, there iS no detectable activity. Values are the mean of three replicates. Vertical bars give standard error where larger than the symbols.

dark. In contrast to DL-usnic acid dehydrogenase, the synthesis of D-usnic acid dehydrogenase is abolished when 100 I~M cycloheximide is included in the incubation media.

Discussion Evernia prunastri contains two usnic acid de- hydrogenases that seem to be produced as a response to very different situations (Fig. 1). One of them, named here DL-usnic acid dehydro-

genase, is removed off from its membranous matrix when the lichen suffers very strong dry- ness under white light as well as nutrient starva- tion (Fig. 2). Solubilization in the cytoplasm of particulate proteins is a very common pheno- menon which affects several catabolic enzymes, such as RNase [7], phosphatases [8] and even reserve proteins [9]. This is due to the loss of membrane water and, according to our results, DL-usnic acid dehydrogenase passes from the membrane to the cytoplasm in a similar way (insert A in Fig. 3). The fact that the highest values of enzyme activity were recovered in a soluble fraction when lichen thalli are irradiated with white light instead of red light is a mere effect of the higher energy content of the first. This is confirmed by the enhanced recovery of dehydrogenase activity when thalli irradiated with low photon flux rate of red light are sub- mitted to high values of osmotic pressure (Fig. 3). There is no evidence of a phytochrome- mediated action of red light on this enzyme since white light can be substituted by PEG treatment and, in addition, active synthesis of protein is not required to the increase of dehydrogenase activity, as deduced from cycloheximide treat- ment (Fig. 2).

We have previously speculated about the re- mobilizing role of this enzyme [1] in order to promote the synthesis of acetyI-CoA from accu- mulated phenol when exogenous nutritional sources fail, in a similar way to that described for flavonoids in higher plants [10]. The environ- mental conditions in which this enzyme activity is enhanced support this previous hypothesis.

The other dehydrogenase is a clearly inducible enzyme, since D-usnic acid dehydrogenase activity appears and increases only when lichen thalli are supplied with an excess of exogenous D-usnic acid over that contained by the lichen itself. In addition, the production of this de- hydrogenase is avoided when the synthesis of proteins is inhibited by cycloheximide. The supplement of exogenous phenol is needed because D-usnic acid behaves as the minor com- ponent in the total phenolic fraction of E. prunastri [11, 12].

Experimental Biological material and culture conditionA Evernia prunastri (L.) Ach., g rowing on branches of Quercus rotundifolia L. and col-

286 CARLOS VlCENTE, AZUCENA GONZALEZ AND MARIA ESTRELLA LEGAZ

lected in Valsain (Segovia, Spain) was used throughout this work. ThaUi were maintained in the dark at 7 ° in polythene bags until required, no more than 15 days.

To assay DL-usnic acid dehydrogenase, samples of 1.0 g of air-dried thalli were maintained in dry conditions and continu- ous darkness, irradiated with white light provided by Sylvania F20TJD 40W fluorescent tubes with a photon flux rate of 25 #mol m -2 s -~ at the level of plants (40% at the PAR) or under red light provided by Sylvania F20T12/R 20W fluorescent tubes with a photon flux rate of 15 p, mol m -2 s -~ (70% at the PAR). Culture temperature was always 26 °. Parallel incubations were carried out by floating thallus samples on distilled water or, when it is indicated, on aqueous solns of polyethylene glycol 600 in red light. Photon flux rate was, in this case, 5 ~,mol m -2 s -1. In addition, a series of samples was infiltrated under pres- sure with 2.0 ml 100 p,M cycloheximide per g of thallus, dried in air f low for 15 min at room temperature and then maintained in white light as above.

To assay D-usnic acid dehydrogenase, 1.0 g of air-dried thal- lus samples were floated on 0.35 mM o-usnic acid in 2% bicar- bonate, pH 8.0, for 14 h in the dark or under white light. When indicated, 100 ~,M cycloheximide was included in the incuba- tion media.

Enzyme assays. Cell-free extracts were prepared by grinding thallus samples in a mortar with 25 ml pure acetone to remove lichen phenolics [13]. The powder was dried in vacuo and then extracted with 15 ml 0.1 M Tris-HCI buffer, pH 8.0, and centri- fuged at --2 ° for 25 min at 27 000 g. When indicated, thalline powder was pre-treated with Triton X-100 for 15 min at 0 ° and then dialysed for 1 h against 2.0 I of the same buffer before centrifugation. Supernatants were used for enzyme assays.

DL-Usnic acid dehydrogenase was assayed by following at 30 ° the rate of enzyme-catalysed oxidation of NADH by L-usnic acid in a reaction mixture containing 0.5 mg protein, 160 l imol Tris-HCI, pH 8.0, 82 nmol L-usnic acid and 3.0 ~,mol NADH in a final volume of 3.0 ml. A unit of specific activity was defined as the decrease in the absorbance at 340 nm per mg protein per min [2]. D-Usnic acid dehydrogenase was assayed in the same way but using D-usnic acid instead of the L-isomer. Protein was

measured by the method of Potty [14] using bovine serum albumin as a standard.

Acknowledgements--This work was supported by a grant from the CAtCYT (Spain) No. 0365 C02 02.

References 1. Vicente, C., Ruiz, J. L. and Estevez, M. P. (1980) Phyton 39,

15. 2. Estevez, M. P., Legaz, M. E., Olmeda, L., Perez, F. J. and

Vicente, C. (1981) Z. Naturforsch. 36¢, 35. 3. Taguchi, H., Sankawa, U. and Shibata, S. (1969) Chem.

Pharm. Bull. 17, 2054. 4. Bandoni, R. J. and Towers, G. H. N. (1967) Can. J. Biochem.

45, 1197. 5. Kutney, J. P., Leman, J. D., Salisbury, P. J., Sanchez, I. H.,

Yee, T. and Bandoni, R. J. (1977) Can. J. Chem. 55, 2336. 6. Kutney, J. P., Baarschers, W. H., Chin, O., Ebizuka, Y., Hur-

ley, L., Leman, J. D., Salisbury, P. J., S&nchez, I. H., Yee, T. and Bandoni, R. (1977) Can. J. Chem. 55, 2930.

7. Marin, B. and Vieira da Silva, J. (1972) Physiol. Plant. 27, 150.

8. Kylin, A. and Quantrano, R. S. (1975) in Plants and Saline Environments (Poljakoff-Mayber, A. and Gale, J., eds) p. 147. Springer, Berlin. Van der Wilden, W., Gilkes, N. R. and Chrispeels, M. J. (1980) Plant Physiol. 66, 390. Barz, W. and Hosel, W. (1975) in The Flavonoids (Harborne, J. B., Mabry, T. J. and Mabry, H., eds) p. 916. Chapman & Hall, London. Blanco, M. J., Suarez, C. and Vicente, C. (1984) Planta 162, 305. Legaz, M. E. and Vicente, C. (1983) Plant Physiol. 71, 300. Vicente, C., Nieto, J. M. and Legaz, M. E. (1983) Physiol. Plant. 58, 325. Potty, V. H. (1969) Analyt. Biochem, 29, 535.

9.

10.

11.

12. 13.

14.