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FEMS Microbiology Letters 238 (2004) 79–84

Elucidation of polysaccharide origin in Ramalina peruviana symbiosis

Lucimara M.C. Cordeiro a, Elfriede Stocker-Worgotter b, Philip A.J. Gorin c,Marcello Iacomini c,*

a Centro de Ciencias Medicas e Farmaceuticas, Universidade Estadual do Oeste do Parana – UNIOESTE, CEP 85819-110, Cascavel, PR, Brazilb Institute of Plant Physiology, University of Salzburg, Hellbrunner Str. 34, A-5020 Salzburg, Austria

c Departamento de Bioquımica e Biologia Molecular, Universidade Federal do Parana, CP 19.046, CEP 81.531-990, Curitiba, PR, Brazil

Received 1 April 2004; accepted 12 July 2004

First published online 23 July 2004

Abstract

A structural elucidation of polysaccharides extracted from the aposymbiotically cultured mycobiont of the lichen Ramalina pe-

ruviana was carried out in order to determine whether the polysaccharides found previously in the symbiotic thalli are produced by

the mycobiont or photobiont or both. The mycobiont isolate was cultivated on a solid malt-yeast extract-medium and the freeze-

dried colonies were defatted and the polysaccharides extracted successively with hot water and aq. 2% KOH, each at 100 �C. Thealkaline extract was obtained in much higher yield (31.5%) and submitted to a freeze-thawing treatment, giving rise to a precipitate

(PK2) of a mixture of (1! 3),(1 ! 4)-a-glucan (1.2:1 ratio, nigeran) and a (1! 3)-b-glucan (laminaran). The mother liquor was

treated with Fehling solution to give a precipitate (galactomannan). This had a (1! 6)-linked a-DD-mannopyranosyl main chain,

substituted at O-4 and in small proportion at O-2,4 by b-Galp units. All three polysaccharides have previously been found in the

symbiotic thalli of R. peruviana, showing that these are produced by the fungus, without the participation of the Trebouxia photo-

biont. Surprisingly, isolichenan, a cold-water soluble (1 ! 3),(1 ! 4)-a-linked-glucan (3:1 ratio) was not found in the isolated my-

cobiont, despite being the main polysaccharide found in the thalli.

� 2004 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.

Keywords: Lichen; Ramalina peruviana; Cultured mycobiont; Polysaccharides; Galactomannan; a- and b-Glucans

1. Introduction

Lichen thalli, the symbiotic phenotype of lichen-

forming fungi in association with their photobiont, are

know to contain considerable amounts of polysaccha-

ride. The lichen Ramalina peruviana contained cold-wa-

ter soluble and insoluble glucans and galactomannan [1].

The soluble a-glucan, isolichenan, is composed by

(1 ! 3) and (1 ! 4)-linkages in a 3:1 ratio, while niger-

an, an a-glucan with a 1:1 ratio of (1! 3) and (1 ! 4)-

0378-1097/$22.00 � 2004 Published by Elsevier B.V. on behalf of the Feder

doi:10.1016/j.femsle.2004.07.020

* Corresponding author. Tel.: +55 41 361 1655; fax: +55 41 266

2042.

E-mail address: iacomini@bio.ufpr.br (M. Iacomini).

linkages, is insoluble in cold water. An insoluble linear

(1 ! 3)-glucan contained b-linkages (laminaran) was al-so present. The galactomannan had a (1! 6)-linked

a-mannopyranosyl main chain, which was substituted

at O-4 and in a small proportion at O-2,4 by b-Galp

units. If these polysaccharides are produced by the my-

cobiont or photobiont alone or by both in symbiosis is

still unknown. However, by comparing extracts of whole

thallus homogenates with those of the aposymbiotically

cultured mycobionts and photobionts, Takahashi et al.[2] demonstrated that most but not all water-soluble

polysaccharides are produced by the fungal partner. Re-

cently, imnunocytochemical location of lichenan within

the thallus of Cetraria islandica using monoclonal anti-

body was reported by Honegger and Haisch [3]. It was

ation of European Microbiological Societies.

80 L.M.C. Cordeiro et al. / FEMS Microbiology Letters 238 (2004) 79–84

found in the extracelullar matrix of the peripheral cortex

and in a thick outer wall layer of medullary hyphae,

while no labelling was recorded on the protoplast of

the myco- and photobiont, and degrading mother cell

wall of the Trebouxia photobiont. Based on their low-

temperature scanning electron microscopy observations,they also suggested that lichenan is a structural, rather

than a storage product, with important functions in

thallus water relations.

We now study the fine chemical structure of polysac-

charides produced by the aposymbiotically grown R. pe-

ruviana mycobiont and compare them with those

produced by the symbiotic lichen in order to fill the gap

in our knowledge of the mycobiont carbohydrates.

2. Materials and methods

2.1. General experimental procedures

Gas liquid chromatography-mass spectrometry (GC-

MS) was performed using a Varian model 3300 gaschromatograph linked to a Finnigan Ion-Trap model

800 mass spectrometer, with He as carrier gas. A capil-

lary column (30 m · 0.25 mm i.d.) of DB-225, held at 50

�C during injection and then programmed at 40

�C min�1 to 220 �C (constant temperature) was used

for quantitative analysis of alditol acetates and partially

O-methylated alditol acetates. Acetylation of alditols

was carried out with Ac2O-pyridine (1:1, v/v) at 100�C for 1 h.

1H and 13C NMR spectra were obtained using a Bru-

ker DRX 400 spectrometer incorporating Fourier trans-

form. Analyses were performed at 50 �C, the water

soluble samples were dissolved in D2O and the water in-

soluble in Me2SO-d6. Chemical shifts are expressed as dPPM, using the resonances of CH3 groups of acetone

internal standard (1H at d 2.224; 13C, d 30.2), orMe2 SOd6(

1H at d 2.60; 13C, d 39.7).

2.2. Isolation and culture of the mycobiont

Sorediate thalli of the lichen R. peruviana (Fig. 1(a))

were collected in the vicinity of Curitiba, Parana, Brazil

at an altitude of 900 m, in August (2001) and were uti-

lized within 2 months of storage at room temperature.Mycobiont cultures were obtained from thallus frag-

ments, following the method of Yamamoto [4] and

grown as described in Cordeiro et al. [5]. For collection

of the mycobiont mass, the colonies (Fig. 1(b)) were ex-

cised with a scalpel from the agar and freeze-dried, to

give 28.0 g of freeze-dried mycelia at the end of a 6

month cultivation. A voucher specimen of the lichen is

deposited in the UPCB (Herbarium of the Federal Uni-versity of Parana), registration number 46.287.

2.3. Extraction and purification of polysaccharides

The mycobiont of R. peruviana (28.0 g) was first ex-

tracted with 2:1 (v/v) CHCl3–MeOH at 60 �C for 2 h

(·3, 500 ml each) and then with 1:1 (v/v) CHCl3–

MeOH at 60 �C for 2 h (·4, 500 ml each), to removelow molecular weight material. The residue was submit-

ted to sequential extraction (Fig. 2) with water at 100

�C for 2 h (·3, 500 ml each) and 2% aq. KOH at 100

�C for 3 h (·4, 500 ml each). The resulting extracts were

neutralised (HOAc), added to ethanol (3 vol.) and the

resulting polysaccharide precipitates were dissolved in

water and dialysed, giving rise to fractions W (aq. ex-

traction) and K2 (alkaline extraction). The K2 solutionwas frozen and then allowed to thaw slowly, and result-

ing insoluble material (fraction PK2) was centrifuged

off. The supernatant SK2 was treated with Fehling so-

lution and the precipitated material (Cu2+-Ppt, galacto-

mannan) centrifuged off. Cu2+-precipitate and

supernatant (Cu2+-Spnt) were neutralised with HOAc,

dialysed against tap water, deionised with mixed ion ex-

change resins, and then freeze-dried. PK2 contained amixture of glucans, which were then suspended in

0.5% aq. NaOH at 50 �C, which dissolved the b-(frac-tion LAM), but not the a-DD-glucans (fraction NIG).

Both fractions were neutralised with acetic acid and di-

alysed.

2.4. Monosaccharide composition of the polysaccharides

Monosaccharide components of the polysaccharides

and their ratios were determined by hydrolysis with 2

M TFA for 8 h at 100 �C, followed by conversion to ald-

itol acetates (GC-MS) by successive NaBH4 reduction

and acetylation with Ac2O-pyridine.

2.5. Determination of homogeneity of polysaccharides and

their molecular weight

The homogeneity and molecular weight (Mw) of wa-

ter-soluble polysaccharides were determined by high

performance steric exclusion chromatography

(HPSEC), using a multidetection equipment in which a

differential refractometer (Waters) and a multiangle la-

ser light scattering apparatus (MALLS; Dawn DSP-F,

Wyatt Technology) were adapted on-line. The eluentwas 0.1 M NaNO3, containing 0.5 g/l NaN3. The poly-

saccharide solution was filtered through a membrane,

with pores of 0.2 lm diameter (Millipore).

2.6. Methylation analysis of polysaccharides

Samples were partially O-methylated using NaOH–

Me2SO–MeI [6]. The per-O-methylated polysaccharides

were converted into partially O-methylated alditol ace-

Fig. 1. Ramalina peruviana: (a) sorediate lichen thallus, bar = 1 cm; and (b) well-developed dark-brown mycelia of the mycobiont, on solid malt-yeast

extract-agar, bar = 5 mm.

Fraction W

Cu++-PptCu++-Spnt

R. peruviana mycobiontFreeze-dried and defatted

Residue

Aq. extraction, 100˚C

Aq. 2% KOH extraction

Galactomannan

Fehling treatment

Supernatant(LAM)

Precipitate(NIG)

Aq. 0.5% NaOH, 50°C

Laminaran Nigeran

Supernatant(SK2)

Residue Fraction K2

Freeze-thawing process

Precipitate(PK2)

Fig. 2. Extraction and purification of polysaccharides of R. peruvi-

ana mycobiont cultivated aposymbiotically on solid malt-yeast

extract-medium.

L.M.C. Cordeiro et al. / FEMS Microbiology Letters 238 (2004) 79–84 81

tates by sucessive treatments with 3% MeOH–HCl for 2

h at 100 �C, 0.5 M H2SO4 for 14 h at 100 �C, reductionwith NaBH4 and acetylation with Ac2O-pyridine. The

products were examined by capillary GC-MS, as de-

scribed in Section 2.1, and identified by their typical

electron impact breakdown profiles and retention times

[7].

3. Results and discussion

In order to remove lipids, pigments and hydrophobic

material, the mycobiont was extracted successively with

CHCl3–MeOH (2:1 and 1:1 ratio, at 60 �C), giving solu-

ble material in a 16.2% combined yield. The defatted

mycobiont mycelia were then submitted to successive

extraction with hot water at 100 �C and aq. 2% KOH

(Fig. 2) and the extracted polysaccharides (fraction W

and K2, respectively) were precipitated with ethanol,dissolved in water and freeze-dried. The fraction K2

was obtained in 31.5% yield, while fraction W showed

low polysaccharide content (5.1%). Fraction K2 was

then submitted to freeze-thawing treatment, resulting

in a high yield of a precipitate (PK2, 18.0% yield) than

the supernatant (SK2, 13.5% yield). In contrast, Corde-

iro et al. [1] observed that when extracted with aq. 2%

KOH, the thalli gave rise to high amounts of solublepolysaccharides (18.2%) and only small ones of insolu-

ble material (1.5%). Honegger and Bartnicki-Garcia [8]

reported that for three cultured mycobionts, most of

the extracted hexosan was alkali-insoluble and account-

ed for almost 40% of the wall dry weight, while alkali-

soluble comprised only 7–15%.

The soluble SK2 fraction contained mannose (52%)

and galactose (37%), with a small proportion of glucose(11%). The polysaccharides present in this fraction were

then separated by treatment with Fehling solution, and

the resulting precipitate (Cu2+-complex, fraction

GMMyc, 8.4% yield) removed by centrifugation. Poly-

saccharides in the supernatant (Cu2+-Spnt) showed

mannose, galactose and glucose in a 25:41:34 ratio,

but as it contained at least three different polymers

(HPSEC) and was formed in a very low yield (1.4%),it was not further investigated.

GMMyc showed mainly mannose (60%) and galac-

tose (40%), being composed of a galactomannan. On

HPSEC analysis, gave a single peak (Fig. 3), showing

a Mw of 43 kD and dn/dc 0.155. According to its 13C

NMR spectrum (Fig. 4(a)), the C-1 signal at low field

of d 103.1 corresponds to non-reducing ends of b-Galp units linked (1! 4) to a-DD-Manp of the main-chain. The C-1 signal at d 100.2 arose from (1!6)-

linked a-DD-Manp units of the main chain substituted

at O-4 by b-Galp. In addition, one small signal ap-

-0.05

0.00

0.05

0.10

0.15

0.20

0 10 20 3 0 40 50

LS, A

UX

(vo

lts)

Volume (mL)

Fig. 3. HPSEC elution profile of water-soluble galactomannan (GMMyc) obtained from R. peruviana mycobiont (refractive index detector).

-D-Galp 1

4

- -D-Manp-(1 6)-4

1-D-Galp

- -D-Manp-(1 6)-

- -D-Manp-(1 6)-2

1-D-Galp

β-D-Galp 1

4

-α-D-Manp-(1→6)-4

1β-D-Gal

-α-D-Manp-(1→6)-

-α-D-Manp-(1→6)-2

1β-D-Galp

A B C

Structure 1: Galactomannan.

82 L.M.C. Cordeiro et al. / FEMS Microbiology Letters 238 (2004) 79–84

peared at d 99.2 which correspond to C-1 of 2,4,6-tri-

O-substituted a-DD-Manp residues. Substitution at OH-

6 of the C-6 of the a-Manp units was also shown on

DEPT examination (Fig. 4(b)), which provided an in-verted signal at d 66.1 corresponding to substituted

CH2OH. Methylation analysis of the galactomannan

indicated non-reducing end units of Galp (42%) and

Manp (3%), as well 6-O (11%), 4,6-di-O (40%) and

2,4,6-tri-O-substituted Manp residues (4%). The

methylation data are consistent with the presence of

a (1 ! 6)-linked a-mannopyranosyl backbone mainly

substituted at O-4 and in small proportion at O-2,4by b-Galp units (Structure 1A, B and C). It follows

that the galactomannan extracted from R. peruviana

mycobiont grown aposymbiotically on a solid malt-

yeast extract-medium has a similar structure to that

9090100100110110

6060

103.

1

100.

2

99.2

66.1

61.1

(a) (b)

Fig. 4. 13C NMR spectra of (a) galactomannan (GMMyc) in D2O, at 50 �Cmycobiont and (b) DEPT experiment.

found previously in the symbiotic thalli [1], only small

differences being observed in the substitution at O-4,

that of the mycobiont galactomannan having 10%

more substitution.

606070708080

75.5

66.1

61.1

, (chemical shifts are expressed as d ppm) obtained from R. peruviana

Fig. 5. 13C NMR spectra of PK2 fraction obtained from R. peruvianamycobiont, in Me2SO-d6, at 50 �C, (chemical shifts are expressed as d ppm): (a)

mixed nigeran and laminaran; (b) purified (1! 3),(1! 4)-linked-a-glucan (1,2:1 ratio); (c) purified (1! 3)-linked b-glucan.

L.M.C. Cordeiro et al. / FEMS Microbiology Letters 238 (2004) 79–84 83

[- α-D-Glcp-(1→3)-α-D-Glcp-(1→4)-]n

Structure 2: Nigeran.

The water-insoluble PK2 fraction was obtained inhigh yield (18.0%) and contained a mixture of

(1 ! 3),(1!4)-linked a-glucan (nigeran) and (1 ! 3)-

linked b-glucan (laminaran), according to its 13C

NMR spectrum (Fig. 5(a)). The glucan mixture was then

suspended in 0.5% aq. NaOH at 50 �C, which dissolved

the b-(fraction LAM), but not the a-linked-glucan (frac-tion NIG). On methylation analysis of the latter, it gave

rise to 2,4,6- and 2,3,6-tri-O-methylglucitol acetates in a

molar ratio of 1.2:1. 13C NMR spectroscopy (Fig. 5(b);

solvent Me2SO-d6) showed high-field C-1 signals at d100.2 and 99.3, characteristic of an a-configuration(Structure 2), with other signals at d 82.5 and 78.9 which

corresponded to O-substituted C-3 and C-4, respective-

ly, and d 60.5 and d 60.1 to unsubstituted C-6, similarto those of a previously described nigeran extracted

from R. peruviana symbiotic thalli [1].

84 L.M.C. Cordeiro et al. / FEMS Microbiology Letters 238 (2004) 79–84

[-β-D-Glcp-(1→3)-]n

Structure 3: Laminaran.

According to its 13C NMR spectrum (Fig. 5(c)), frac-tion LAM consists of a (1 ! 3)-linked b-glucan (Struc-

ture 3), with a low-field C-1 signal at d 103.1, and

others at d 86.3 (O-substituted C-3), d 76.5 (C-5), d73.1 (C-2), d 68.6 (C-4) and 61.0 (C-6). This b-glucanhad also been extracted from the symbiotic thalli of

R. peruviana [1] and its isolation from the aposymbioti-

cally cultured mycobiont confirms its fungical origin.

However, for many years some authors had suggesteda photobiont origin, due its low yield when obtained

from lichen thalli and by its similar structure to algal

laminarans [9–13].

Surprisingly, isolichenan, a cold-water soluble

a-(1 ! 3), (1 ! 4)-linked-glucan (3:1 ratio) was not

found in the W (data not shown) and K2 extracts of

the aposymbiotically cultured R. peruviana mycobiont.

On the other hand, it was the main polysaccharide ex-tracted from the symbiotic thalli (20.7% yield), suggest-

ing that this soluble glucan is produced by the

mycobiont, only in the presence of a photobiont. What

triggers this phenomenon is still unknown. To verify the

hypothesis that isolichenan suppression is influenced by

the culture medium, other carbon and nitrogen sources

should be screened in future experiments. We do not be-

lieve that isolichenan has a photobiont origin. Recently,Honegger and Haisch [3] using a monoclonal antibody,

located a b-glucan (lichenan) in the extracellular matrix

of the peripheral cortex and in a thick outer wall layer of

medullary hyphae, while no labelling was recorded on

the protoplast of the myco- and photobiont and on

the mother cell wall of Trebouxia photobiont.

In conclusion, our studies with isolated polysaccha-

rides from the aposymbiotically cultured R. peruviana

mycobiont showed that the galactomannan, the

a-(1 ! 3),(1 ! 4)-glucan (1:1 ratio) and the b-(1 ! 3)-

glucan found in the symbiotic thalli are produced by

the fungus, without the participation of the Trebouxia

photobiont. A question is still open, namely, does the

photobiont of R. peruviana give any contribution to

the polysaccharide content of the symbiotic thallus?

Further studies are needed to resolve this question.

Acknowledgements

The authors thank Dr. Marcello Pinto Marcelli (Bo-

tanical Institute, Sao Paulo, Brazil) for identification of

the lichen. CAPES and PRONEX-Carboidratos provid-

ed financial support, without which this investigation

would not have been possible. E-STWO is very grateful

to the Austrian Science Foundation (FWF) for gener-

ously supporting the cooperation between Brazil and

Austria with the help of Grant 15328-BIO.

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