obaria pulmonaria is a large foliose lichen specieswhich was common in pre-industrial periods in hu-mid temperate and boreal regions of the Northern
hemisphere (SCHEIDEGGER, FREY & WALSER 1998). Unfortu-nately, it is actually in strong decline in most European coun-tries (RICHARDSON 1992, SCHEIDEGGER 1995, SCHEIDEGGER,FREY & ZOLLER 1995).
The growth pattern of Lobaria pulmonaria was describedas geotropically differentiated into upwards and downwardsgrowing lobes (SCHEIDEGGER, FREY & WALSER 1998).
This pattern derives from a triplication of the apical meris-tem of young lobes, leading to one meristem growing upwards,one horizontally and one growing downwards.
Initially, apical meristem of lobes growing downwards isactive and these lobes have an increased growth rate. But la-ter, the formation of vegetative diaspores, at the margins oflobes growing downwards, inactivates their apical meristems,inhibiting the longitudinal growth. These lobes become senile(SCHEIDEGGER, FREY & WALSER 1998). Upwards meristema-tic lobes develope according to a upright direction. They showa continuous growth, because their apical meristem is not in-activated by the formation of vegetative diaspores.
The observations reported above are at the base of the pre-sent research, conceived to exploring this pattern of growth ina lichen and to verify whether the inactivation of meristems
in downwards senile lobes of L. pulmonaria, due to the forma-tion of vegetative diaspores leads to any anatomical, histo-chemical and cytological differentiation, in comparison toupwards young meristematic lobes. As will be discussed inthis paper the use of a simple and sensitive histochemicalprocedure, the Toluidine Blue O test, extensively used in ourresearch team (MODENESI & VANZO 1986, MODENESI et al.2000) for its usefulness in lichen histochemistry, has revealeda clear differentiation between the two types of lobes, provi-ding information of their different nutritional status.
Materials and methods
Microscopical investigation were carried out on the upwardsgrowing meristematic and downwards growing sorediate thal-line lobes of five individual thalli of Lobaria pulmonaria (L.)Hoffm., collected on the bark of chestnuts trees in Aveto valley(Liguria, North-western Italy) 1000 m above the sea level.Small apical portions of both lobe types (about 0.5–1 cm) werefixed in FAA (formalin, acetic acid, ethanol 60 %) (SASS
1958) dehydrated in an ethanol series and embedded in JB4,a glycol methacrylate resin (Conventional JB4 procedure, Poly-science, Inc. Warrington). In the latter preparation, specimenswere fixed in 3 % glutaraldehyde in 0.1 M cacodylate bufferat pH 6.8 for 2 h at 0–4 °C (O’BRIEN & MCCULLY 1981), de-hydrated and embedded with three changes of cold (0–4 °C)JB4 resin (Cold JB4 procedure). Exact orientation of samplesand serial sectioning from distal to proximal portions of lobeswere allowed by embedding in bottle-neck polyethylene cap-sules (Polyscience Inc., Warrington). 5–10 µm thick sections
Anatomical and histochemical differentiation in lobes of the lichenLobaria pulmonaria
Paolo GIORDANI 1*, Giorgio BRUNIALTI1
The growth pattern of the thallus of Lobaria pulmonaria, a foliose lichen, has been described as differentiated into upwardsand downwards growing lobes. The former show meristematic properties, whereas the latter, owing to the formation ofsoralia inactivating apical meristems, become senile lobes. A simple and sensitive histochemical procedure, the TBO testperformed in this study, shows co-distribution of zones of active growth and polyphosphates accumulation in L. pulmo-naria and gives evidence of the central role of phosphate in lichen metabolism. Upwards and downwards growing lobesmainly differentiate in pattern of accumulation of histochemically detectable polyphosphates. In the former, actively growingzones correspond to the algae, cortical and medullary hyphal cells adjacent to the algal layer of the pseudomeristematic mar-ginal rim and of the adjacent elongation zone. In the downwards sorediate lobes, lacking apical growth, the actively growingzones correspond to medullary hyphae and algae occurring in areas where soredia are formed.
Mycological Progress 1(2): 131–136, 2002 131
1 Dipartimento per lo Studio del Territorio e delle sue Risorse – Sededi Botanica. Corso Dogali 1M, 16136 Genova, Italy. E-mail: [email protected]
were cut with a glass knife on a Reichert OM2 microtome.The following tests were performed on sections: 1) ToluidineBlue O (TBO) (Sigma), 0.05 % in acetate buffer at pH 4.4(O’BRIEN & MCCULLY 1981) and at pH 1, by adjustment with6N HCl (LING-LEE, ASHFORD & CHILVERS 1977), for 1 min,as general and histochemical stain. 2) Trichloroacetic acid(TCA) extraction: sections were pre-treated with 10% TCAprior to staining with TBO at pH 1 to show the differentialpolyanions lability (ASHFORD, LING-LEE & CHILVERS 1975).3) Alcian Blue 8GX (Sigma) 1 % at pH 2.5 in 3 % acetic acidand at pH 0.5 in 0.2 N HCl for 30 min for acid polysaccha-rides (LEV & SPICER 1964). 4) Periodic-acid-Shiff (PAS)procedure was used for general polysaccharide localisation(FEDER & O’BRIEN 1968). 5) Autofluorescence: embeddedand unstained sections were observed under a Leitz Dialuxepifluorescence microscope equipped with an HBO 50 Wmercury vapour lamp and an H2 Leitz filter block (BP 420–490).
Results
The observations refer to two different types of thalline lobesamples of L. pulmonaria: upwards growing young meriste-matic (UM) lobes, downwards growing senile sorediate (DS)lobes.
UM lobes of L. pulmonaria, as visible by cross sectionsobserved in fluorescence and light microscopy, showed dif-ferent thalline areas as one move from the tips of the thallusmargin to the distal thalline zones. Like the majorities of fo-liose, dorsiventrally-organized, heteromerous thalli withmarginal growth (HONEGGER 1996), three zones can be dis-tinguished in sequence from the tip: a pseudomeristematicmarginal rim, an elongation area and a fully differentiated sub-apical area, or mature area. A further zone, localised beforethe marginal rim, is visible. This zone was formed by myco-biont cells only and organised in two layers: an external,faintly yellow-brown pigmented in unstained sections andfluorescing yellow when observed under blue exciting light
(Fig. 1A) and an internal one, dark pigmented in unstainedsections and non-fluorescing under the same condition of ex-citing light (Fig. 1A). Both layers reacted positively with PAStest, showing a polysaccharidic composition (FEDER & O’BRIEN
1968). The inner dark-pigmented layer showed the hystoche-mical reactivity of phenolic substances (LING-LEE, ASHFORD
& CHILVERS 1977) staining blue-green with TBO. In severalsections (Fig. 1C) the green colour was masked by the extentof oxidized phenolic substances and showed a brownish-greencolour. The external layer showed the ortochromatic (blue)reaction of TBO, in relation to the accumulation of the dye.
The pseudomeristematic marginal rim showed the ana-tomical peculiarities of a pseudomeristem (HILL 1985, 1989,1992) with dense, small mycobiont and photobiont cells andwith high cell turnover rates in both the partners (Fig. 1A, C).
Sections tested with TBO at pH 4.4 revealed in this zonenumerous granules stained vivid red, the γ-metachromatic re-action, and ranged downwards in size from 1 µm (Fig. 1C, G).These granules were found to be concentrated mainly in thealgal cells, in the medullary hyphae immediately in contact tothe algal cells and in the upper cortical hyphae. Red granuleswere never observed in the medullary hyphae outside ofcontact of algal cells. The fungal cytoplasm and the apoplas-tic compartment (cell wall and interhyphal matrix) of the up-per cortex also gave a metachromatic reaction with TBO atpH 4.4, pale in the former and strong in the latter, but this wasa purplish colour (β-metachromasy). However, when sectionswere stained with TBO at pH 1 or were rinsed briefly with 0.1N HCl after staining with TBO at pH 4.4, only the vivid redstaining of the metachromatic granules remained. Red colourdisappeared when sections were treated with cold TCA beforestaining with TBO, in contrast to unaffected β-metachromasywhich persisted. Granules did not stain with Alcian Blue andPAS, showing a non polysaccharidic composition in contrastto the apoplastic compartment which reacted positively to boththe tests (LING-LEE, ASHFORD & CHILVERS 1977, CHILVERS,LING-LEE & ASHFORD 1978).
In the elongation thalline zone, approximately 0.15-0.2 mmbehind the pseudomeristematic tip, the cells of both partners
132 Anatomical and histochemical features of Lobaria pulmonaria
Fig. 1. Cross-sections of the thallus of Lobaria pulmonaria. A-B, autofluorescence of resin embedded sections under blue lightexcitation. A, upwards growing meristematic lobe. In the lobe tip are visible a fluorescing yellow external layer (e), a non-fluorescing internal one (i) and a pseudomeristematic marginal rim (p) showing a high algal cell turnover rate. B, downwardsgrowing sorediate lobe, lacking the pseudomeristematic marginal rim and the internal layer visible in A. No cell turnover is evi-dent. C-G, light microscopy sections stained with TBO. C, upwards growing meristematic lobe showing an external layer (e),an internal brownish-green stained one (i) and a pseudomeristematic marginal rim (p) with high cell turnover. Polyphosphategranules (arrow) occur both in algal and fungal cells. D, downwards growing sorediate lobe, lacking the pseudomeristematicmarginal rim and the internal layer. No polyphosphate granules are visible. E, upper cortex and algal layer of the fully diffe-rentiated area. No polyphosphate granules and a very limited cell turnover occur. F, developping soredium in downwardsgrowing lobe. Arrow points out the occurence of polyphosphate granules in an algal cell. G, upwards growing meristematiclobe. Upper cortex and algal layer in thalline area placed between the pseudomeristematic marginal rim and the elongation zone.Polyphosphate granules (arrows) occur in medullary and cortical hyphae in contact with algal cells. Bars: 25 µm.
gain their mature dimension and, progressively, showed adecreasing cell turnover.
A very limited cell turnover can be observed in the fullydifferentiated areas or non-growing areas (HONEGGER 1993),approximately 10 mm behind the elongation zone. No γ-meta-chromatic granules were observed in this mature zone (Fig.1E).
In DS lobes, pseudomeristematic marginal rim and elon-gation area were lacking (Fig. 1B, D), only an area showingthe above described features of the fully differentiated zonewas visible until the tip of thalline lobe. TBO γ-metachroma-tic reaction was always negative.
In these lobes high cell turnover rates in the fungal and al-gal partners were visible in zone where soredia are developing(Fig. 1F). In these zones γ-metachromatic granules were ob-served in medullary hyphal and algal cells.
Discussion
Presented results show that UM and DS lobes of L. pulmona-ria differentiate in pattern of accumulation of histochemicallydetectable red granules in thalline compartments characteri-sed by active algal and fungal growth.
The red reaction (γ-metachromatic) of the granules stainedwith TBO, was readily distinguishable from the β-metachro-matic (purple) reaction of the upper cortical apoplastic com-partment, probably due to an acid polysaccharidic (mucopoly-saccharidic) component (CHILVERS, LING-LEE & ASHFORD
1978, MODENESI & VANZO 1986).Red staining, persistence of staining at pH 1 and TCA labi-
lity provide evidence that the granules contain polyphospha-tes as a major constituent (ASHFORD, LING-LEE & CHILVERS
1975).Staining properties of granules observed in L. pulmonaria
are identical to polyphosphates granules (also termed volutin)observed by CHILVERS, LING-LEE & ASHFORD (1978) in algaland fungal partners of Collema leucocarpon and Peltigeradolichorrhiza, by MODENESI & LAJOLO (1987) in excipularhyphae of the foliicolous lichen Fellhanera bouteillei (subCatillaria bouteillei), and in mycorrhizas of various plants(LING-LEE, CHILVERS & ASHFORD 1975, LING-LEE, ASHFORD
& CHILVERS 1977, CALLOW et al 1978).In UM lobes the actively growing zones, accumulating
polyphosphates, correspond to the cortical hyphae, algae andmedullary hyphal cells, adjacent to the algal layer of the pseu-domeristematic marginal rim and of the adjacent elongationzone. In the DS lobes, lacking apical growth, the activelygrowing zones correspond to medullary hyphae and algae oc-curring in areas where soredia are formed. Our results agreewith the observations of SCHEIDEGGER, FREY & WALSER
(1998) which define upwards young lobes as continuouslygrowing (meristematic) and downwards sorediate ones as se-nile lobes, in which meristems are inactivated by formationof soredia.
Co-distribution of zones of active growth and polyphos-phates accumulation in L. pulmonaria well gives evidence ofthe central role of phosphate in lichen metabolism (FARRAR
1976).Polyphosphates isolated from bacteria, algae and fungi
(KULAEV & VAGABOV 1983) are linear condensed polymersof inorganic phosphate (Pi) in which orthophosphate residuesare linked through energy-rich phospho-anhydride bondssimilar to those connecting terminal phosphate residues in themolecule of ATP and other nucleoside oligophosphates(KULAEV 1975). These compounds and the energy that can bereleased during their hydrolysis are involved in dynamic P sto-rage, growth control, synthesis of proteins, nucleic acids andphospholipids and cell division (HAROLD 1966). Polyphosha-tes may be broken down by enzymes of the polyphosphatekinase type (producing ATP) or poliphosphatase type (libe-rating Pi) and in direct-phosphorylation of sugars by a poly-phosphate glucokinase action (HAROLD 1966).
Polyphosphates are able to sequester and release cations,i.e. Ca, Mg, K, which in turn may regulate activity of variousenzymes and they are also involved in cellular detoxificationby complexing metals (HASHEMI, LEPPARD & KUSHNER 1994).
According to FARRAR (1976) the rainwater, containing lowconcentrations of minerals (NEWMAN 1995) is the main sour-ce of nutrients, such as phosphorus, for many lichens. CRIT-TENDEN, KALUKA & OLIVER (1994) argued that lichen growthmay be limited by the rate of income from the atmosphere andtherefore internal conservation of Pi may be crucial for the sur-vival and growth (HYVARINEN & CRITTENDEN 2000).
In Hypogymnia physodes (FARRAR 1976) the pattern ofmetabolism of absorbed Pi from bathing solutions, is domi-nated by the high percentage of Pi that is not metabolised, butrapidly incorporated in an insoluble fraction which was ten-tatively identified as polyphosphates. FARRAR (1976) hypo-thesised a double significance for polyphosphates in lichens,a store of Pi which can be utilised when environmental suppliesfall and a mean of storing a scarce anion in a non-leaking form,due to considerable leakage of Pi which occur on rewettingdry lichens (FARRAR & SMITH 1976).
Polyphosphates accumulation in UM lobes suggests an in-teresting parallel to growing apically and degenerating basalthalline parts (mat-forming) lichens of the genus Cladonia(subgenus Cladina), Stereocaulon and Cetraria. In these lichensthe occurrence of maximum concentration of N, P and K inthe apices leads to speculate that demand for nutrients in thesegrowing zones might be partially satisfied by upwards trans-location or resources from degenerating bases in a source-sinkrelationship (HYVARINEN & CRITTENDEN 1998, 2000). Thisview is supported by a recent work (HYVARINEN & CRITTEN-DEN 2000) demonstrating that P is recycled within podetia ofCladonia portentosa, a mat-forming lichen, migrating fromdegrading basal regions upwards to the growing apices follo-wing a source-sink relationship.
We can hypothesise a similar relationship in L. pulmonariathallus, in which sinks may be represented by actively growing
134 Anatomical and histochemical features of Lobaria pulmonaria
zones: pseudomeristematic marginal rim and elongation zoneof UM lobes and areas forming soredia in DS lobes. In con-sequence, sources may be the fully differentiated or the ma-ture thalline proximal zones, behind the apical parts, for theformer and areas bordering the forming soredia zone for thelatter.
The current view of phosphorus transport along hyphae ofmychorrizal mycelia is that it occurs largely by transport oforthophosphate, either by diffusion, osmotically generatedmass flow, or cytoplasmic streaming and also via movementof small polyphosphate granules, presumably in vacuoles, alsocarried by cytoplasmic streaming (ORLOVICH & ASHFORD
1993). We can presume some similarities of transport me-chanisms, on the basis of no being fundamental differences atthe cytological level between lichenized and non lichenizedfungi (HONEGGER 1996, HYVARINEN & CRITTENDEN 2000).
On the other hand, the role of mycorrhizal fungi in en-hancing the phosphorus status of their higher plant associatedis well documented (HARLEY & SMITH 1983). This is thoughtto be achieved by absorption of Pi from the soil by extrama-trical hyphae and its translocation through the mycelium tothe root surface. In lichens, FARRAR (1976) argued that Pi ab-sorption must be largely due to the fungus, not being evidenceif Pi enters the algae directly. Data presented here show redgranules in algae, but does not contribute to resolve this ques-tion.
Another feature differentiating UM and DS lobes is thedouble layers bordering apical tips occurring in the formerand absent in the latter. This feature may be related to the cha-racteristic of growing zones of UM lobes. According to HO-NEGGER (1993), the first recognisable event, in a stratifiedthallus differentiation, is the secretion of extracellular gelati-nous material, filling the space between cells which may beinterconnected by anastomoses. After this, a peripheral con-glutinate zone, the pseudoparenchymatous cortex, is formed.A defensive role to protect the meristematic zone may be hy-pothesised for the phenolic component, occurring in the innerlayer and showed by green TBO reaction (RUNDEL 1978).
Acknowledgements
The authors are deeply indepted with Paolo Modenesi (Ge-nova) and Christoph Scheidegger (Birmensdorf) for usefulcomments. The helpful suggestions of the anonymous revie-wers on the manuscript are greatly appreciated.
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