The lamellar cells of winter leaves of Polytrichum formosum were studied by electron microscopy of thin sections, and the results were compared with those for spring and summer plants. Pigment content and photosynthetic activity were also analysed. The lamellar cells of winter plants, when grown at temperatures above zero, had a similar ultrastructure to those of spring and summer plants. Their pigment content and photosynthetic activity were also similar. On the other hand, the lamellar cells of frozen plants (examined at temperatures
about –5 °C) were desiccated, i.e. deformed, the cytosol was shrunken, and the ribosomes and mitochondria were aggregated. The thylakoid system of their chloroplasts was irregularly arranged and the grana thylakoids were compressed. The desiccation of the cells was soon annulled when the ambient temperature rose above zero

M. Wrischer

N. Ljubešić

T. Prebeg

Z. Devidé

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Acta Bot. Croat. 64 (2), 219–226, 2005 CODEN: ABCRA25ISSN 0365–0588Structural changes of lamellar cells in leaves of themoss Polytrichum formosum Hedw. during winterfreezing and thawing processesNIKOLA LJUBE[I]*, MERCEDES WRISCHER, TATJANA PREBEG, ZVONIMIR DEVIDÉRu|er Bo{kovi} Institute, Department of Molecular Biology, P.O. Box 180, HR-10002Zagreb, CroatiaThe lamellar cells of winter leaves of Polytrichum formosum were studied by electron mi-croscopy of thin sections, and the results were compared with those for spring and sum-mer plants. Pigment content and photosynthetic activity were also analysed. The lamellarcells of winter plants, when grown at temperatures above zero, had a similar ultrastructureto those of spring and summer plants. Their pigment content and photosynthetic activitywere also similar. On the other hand, the lamellar cells of frozen plants (examined at tem-peratures about –5 °C) were desiccated, i.e. deformed, the cytosol was shrunken, and theribosomes and mitochondria were aggregated. The thylakoid system of their chloroplastswas irregularly arranged and the grana thylakoids were compressed. The desiccation ofthe cells was soon annulled when the ambient temperature rose above zero.Key words: Polytrichum formosum, leaves, ultrastructure, pigment, photosynthesis, la-mellar cell, freezing, thawingIntroductionThe moss species that grow in temperate climatic zones (at medium geographic lati-tudes) are well adapted to low winter temperatures, and are, at higher sea levels, even inearly spring and late autumn, exposed to daily freezings (at night) and thawings (during theday). During the winter months they may remain at subzero temperatures for days, or evenweeks, and are then frequently covered with snow or ice.Numerous studies on overwintering higher plants have indicated that, during autumnand early winter, these plants undergo acclimation to low temperatures. This process com-prises a variety of structural and biochemical adaptations, including changes in thevacuolar system, increase of carbohydrate levels, shifts in the lipid composition of thebiomembranes, and de novo synthesis of specific proteins (SENSER and BACK 1984, SAUTERet al. 1996, UEMURA and STEPONKUS 1997, ROUTABOUL et al. 2000, ATICI and NALBAN-TOGLOU 2003). At present, the expression of the genes responsible for these changes is re-ceiving particular attention (BROWSE and XIN 2001, THOMASHOW 2001, SUNG et al. 2003).ACTA BOT. CROAT. 64 (2), 2005 219* Corresponding author: Fax +385 1 45 61 177, e-mail: ljubesic@irb.hrDuring the freezing process, ice crystals that are formed outside the cells, act as nucleationsites for water, which is thus pulled out of the cells. This eventually results in desiccation ofthese cells (PEARCE and ASHWORTH 1992, WRISCHER et al. 2000, BALL et al. 2004). We as-sumed that similar mechanisms may also function in mosses exposed to subzero tempera-tures.The anatomy of the leaves of the large moss, Polytrichum formosum, is structurallycomplex. The abaxial part of the leaf consists of large cells with thick walls, the hydroids,which function in the transport of water, and of leptoids, which are efficient in the transportof sugars (THOMAS et al.1990). The closely arranged longitudinal rows of one cell-layerwide chlorophyll-containing, lamellae on the adaxial side of the leaf have the same func-tion as the mesophyll in higher plants (PAOLILLO and REIGHARD 1967, THOMAS et al. 1996).The lamellar cells are directly exposed to the environment. We studied how low wintertemperatures influence the structure and physiology of these cells in Polytrichum.Materials and MethodsSamples of Polytrichum formosum Hedw. were collected at its natural habitat, in theMedvednica mountains (near Zagreb), at an altitude of 600–1000 m above sea level. Forelectron microscopy, the plants were immediately fixed at their habitat; for pigment analy-ses and measurements of photosynthesis and respiration, they were transferred to the labo-ratory. The fixation medium (both for light and for electron microscopy) consisted of 1%glutaraldehyde in 0.05 M cacodylate buffer (pH 7.2). The duration of the fixation was 1–2h, and the temperature of the fixation medium about +2 °C, except when lower tempera-tures – for the fixation of frozen objects (about –5 °C) were needed. In these cases the vialswith the fixation medium were thoroughly cooled by dipping them into the snow/ice thatsurrounded the moss plants. After rinsing in cold buffer, the material was postfixed in 1%OsO4, for 2 h. The samples were then dehydrated in a series of ethanol and in acetone, andembedded in Spurr’s medium. For light microscopy, semithin sections were stained withtoluidine blue and examined under a light microscope (Zeiss Axiovert 35). For electron mi-croscopy, the thin sections were stained with uranyl acetate and lead citrate and examinedusing a Zeiss EM 10 or a FEI Morgagni 268D microscope.Pigments were extracted in 80% acetone. Their concentration was calculated accordingto LICHTENTHALER (1987). Respiration and photosynthetic activity (production of oxygen)were measured with an oxygen electrode (Hansatech Ltd., London) at 20 °C. The reactionmixture contained 0.1 mol/L K-Na-phosphate buffer (pH 7.2) and 0.01 mol/L Na-bicarbon-ate. The samples for photosynthetic measurements were illuminated with a halogen lampand, for respiration measurements, they were kept in the dark (WRISCHER at al. 1998,LJUBE[I] et al. 2003).ResultsThe arrangement of the cells in the lamellae on the adaxial side of Polytrichum leaveswas best visible by light microscopy when the leaves were cut perpendicularly to their longaxis. In spring leaves, the peripherally arranged chloroplasts were well visible in the round-ish lamellar cells (Fig. 1). In winter, these cells remained roundish, but were filled with a220 ACTA BOT. CROAT. 64 (2), 2005LJUBE[I] N., WRISCHER M., PREBEG T., DEVIDÉ Z.dense material, and the marginal cells became intensely stained (Fig. 2). The frozen cells ofmaterial collected in winter (at –5 °C) had conspicuously irregular outlines (Fig. 3).Electron microscopy revealed that the lamellar cells were covered by a thick cell wall,which was particularly thick on the marginal cells. In spring plants, several lightly stainedvacuoles surrounded the centrally arranged nucleus (Fig. 4). The large chloroplasts (3–5per cell section) contained loosely arranged grana of 3–5 thylakoids, connected to singlestroma (intergrana) thylakoids. In the stroma, there were several starch grains and groupsof plastoglobules, 0.1 to 0.15 µm in diameter. Small mitochondria, peroxisomes, elementsof the endoplasmic reticulum and some dictyosomes were dispersed in the cytoplasm (Fig.5). Some, about 0.5 µm large, lipid globules were usually located near the cell wall. Thelamellar cells of the summer leaves were organized in a similar way. Large chloroplasts indivision were often found in these leaves.In winter leaves, the vacuoles of the marginal lamellar cells were filled with a densegranular material (Fig. 6). The other cell organelles were similarly organized as in spring orsummer leaves. The chloroplast stroma contained starch grains and plastoglobules with anapproximate diameter of 0.1 µm (Fig. 7).The structure of frozen winter leaves differed substantially from that of unfrozen ones.In the lamellar cells, the volume of the cytosol had shrunk and the ribosomes were aggre-gated and assembled in the areas neighboring the vacuoles. The mitochondria were alsopacked in between the vacuoles, while the endoplasmic reticulum and the dictyosomeswere not discernible (Fig. 9). In the large, irregularly shaped, chloroplasts the thylakoidsystem was irregularly arranged and its membranes were wavy. The grana thylakoids to-gether with their lumina were compressed (Figs. 8, 10, 11). The peripheral part of thechloroplast stroma was without thylakoids, but numerous straight invaginations of the in-ner membrane of the envelope became visible in these regions. In the stroma, plas-toglobules were numerous and starch grains were still present (Figs. 8, 10).ACTA BOT. CROAT. 64 (2), 2005 221MOSS LEAVES DURING WINTER FREEZING AND THAWINGFigs. 1–3. Semithin sections through lamellar cells. Fig. 1. Cells from a spring plant. Fig. 2. Cellsfrom a winter plant collected at a temperature above zero. The marginal cells are intenselystained. Fig. 3. Cells from a frozen winter plant collected at an ambient temperature of –5°C. The cells have indentations (arrowheads). Bars = 20 µm.Only a short temperature increase above zero was sufficient to reverse the effect of freezing.The cells became roundish again, the cytosol reassumed its normal structure, and in thechloroplasts, the grana thylakoids returned to their regular, loosely arranged, pattern (Fig. 12).Pigments and photosynthetic activity were measured several times during spring, sum-mer, autumn and winter. The chlorophyll and carotenoid levels did not change much,throughout the year. The values found for the photosynthetic activity of winter leaves wereonly slightly lower than those found in spring and summer leaves (Tab. 1).222 ACTA BOT. CROAT. 64 (2), 2005LJUBE[I] N., WRISCHER M., PREBEG T., DEVIDÉ Z.Figs. 4–5. Thin sections through lamellar cells in spring (April–May). Fig. 4. The marginal cellshave thick cell walls. Bar = 5 µm. Fig. 5. A lamellar cell with chloroplasts, mitochondria(m), and several vacuoles (v). g = lipid globules. Bar = 1µm.DiscussionPolytrichum formosum tolerates low winter temperatures excellently. The ultrastruc-ture, the chlorophyll content and the photosynthetic activity of the lamellar cells, in winter,remained similar to those in spring and summer plants. Low night temperatures, during au-tumn, may already stimulate acclimation to low temperatures. In mid winter, the plants hadalready passed through repeated freezings and thawings and were well-acclimated to freez-ing temperatures. Our ultrastructural analyses showed that freezing the cells resulted indesiccation (dehydration). By applying cryo-scanning electron microscopy some authorsdetected desiccation and ice crystals in the intercellular spaces of frozen higher-plantleaves (PEARCE and ASHWORTH 1992, BALL et al. 2004). With our method of sample prepa-ration, the deformed shape of the frozen moss cells and the collapse of the cell lumen un-equivocally indicated their desiccation. There appears to be a genetically defined overlap-ping relationship between cold acclimation and dehydration (BROWSE and XIN 2001). It isassumed that the natural desiccation tolerance of mosses also explains the excellent resultsachieved with cryopreservation (BURCH 2003).The desiccation in frozen plants of Polytrichum could, within a surprisingly short pe-riod of time, be annulled by exposure to above-zero temperatures. This could even happenACTA BOT. CROAT. 64 (2), 2005 223MOSS LEAVES DURING WINTER FREEZING AND THAWINGFigs. 6–7. Thin sections through lamellar cells of material collected in winter at ambient tempera-tures above zero. v = vacuoles, m = mitochondria, n = nucleus, s = starch, d = dictyosome.Fig. 6. The vacuoles of the marginal cell are filled with a granular material. Fig. 7. Thethylakoid system of the chloroplasts has a normal ultrastructure. There are starch grainsand plastoglobules in the chloroplast stroma. Bars = 1µm.during the preparation (fixation) of the tissue, when the temperature of the fixation mediumwas slightly higher (i.e. slightly above zero) than that of the frozen plant.The unusual membraneous invaginations of the inner chloroplast membrane have beenfound repeatedly in frozen chloroplasts, during the winter months (SENSER and BECK 1984,224 ACTA BOT. CROAT. 64 (2), 2005LJUBE[I] N., WRISCHER M., PREBEG T., DEVIDÉ Z.Figs. 8–10.Thin sections through the lamellar cells of frozen material collected during winter, at anambient temperature of –5 °C. n = nucleus, m = mitochondrion, v = vacuoles, g = lipidglobule, arrowheads = invaginations of the inner chloroplast membrane, arrows = aggre-gated ribosomes. Fig. 8. Irregularly shaped chloroplasts with enlarged stroma. Fig. 9. Partof a cell with ribosomes compressed between the vacuoles. Fig. 10. Part of a chloroplastwith compressed grana thylakoids and starch grains in the stroma. Bars = 1µm.WRISCHER et al. 2000, LJUBE[I] et al. 2003). The increased thylakoid-free stroma regions infrozen Polytrichum lamellar cells facilitated the detection of these invaginations. Whatcauses these structures is still unknown. A possible explanation would be a different lipidcomposition of the inner and the outer chloroplast membranes causing an unequal responseto low temperatures (BROWSE and XIN 2001).AcknowledgementThe authors would like to thank Dr. Volker Magnus for the critical reading of the manu-script.ReferencesATICI, Ö., NALBANTOGLOU, B, 2003: Antifreeze proteins in higher plants. Phytochemistry64, 1187–1196.BALL, M.C., CANNY, M. J., HUANG, C. X., HEADY, R. D., 2004: Structural changes inacclimated and unacclimated leaves during freezing and thawing. Funct. Plant Biol. 31,29–40.BROWSE, J., XIN Z., 2001: Temperature sensing and cold acclimation. Curr. Opin. PlantBiol. 4, 241–146.BURCH, J., 2003: Some mosses survive cryopreservation without prior pretreatment. Bry-ologist 106, 270–277.ACTA BOT. CROAT. 64 (2), 2005 225MOSS LEAVES DURING WINTER FREEZING AND THAWINGFigs. 11–12. Chloroplast grana of winter leaves at high magnification. Fig. 11. Compressed granathylakoids from a frozen leaf (–5 °C). Fig. 12. Grana thylakoids from thawn / defrozenleaf (above zero). Bars = 0.2 µm.Tab. 1. Total chlorophylls, total carotenoids, photosynthetic activity and respiration of Polytrichumleaves.Total chlorophylls(g/g fr. wt.)Total carotenoids(g/g fr. wt.)Photosynthetic activity(mol O2/g fr.wt./h)Respiration(mol O2/g fr.wt./h)spring 2.530.11 0.550.05 125.124.88 40.285winter 2.170.02 0.470.05 115.1314.2 45.834.17LICHTENTHALER, H. K., 1987: Chlorophylls and carotenoids. Pigments of photosyntheticmembranes. Meth. Enzymol. 148, 350–382.LJUBE[I], N., WRISCHER, M., PREBEG, T., DEVIDÉ, Z., 2003: Chloroplast structure and func-tion in wild-type and aurea-type leaves of the Japanese spindle-tree over their life span.Acta Bot. Croat. 62, 1–10.PAOLILLO, D. J. JR., REIGHHARD, J. A., 1967: Ultrastructural features of some poly-trichaceous moss leaves. Bryologist 70, 61–69.PEARCE, R. S., ASHWORTH, E. N., 1992: Cell shape and localization of ice in leaves ofoverwintering wheat during frost stress in the field. Planta 188, 324–331.ROUTABOUL, J.-M., FISCHER, S. F., BROWSE, J., 2000: Trienoic fatty acids are required tomaintain chloroplast function at low temperatures. Plant Physiol. 124, 1697–1705.SAUTER, J. J., WISNIEWSKI, M., WITT, W., 1996: Interrelationships between ultrastructure,sugar levels, and frost hardiness of ray parenchyma cells during frost acclimation anddeacclimation in poplar (Populus x canadensis Moench "robusta") wood. J. PlantPhysiol. 149, 451–461.SENSER, M., BECK, E., 1984: Correlation of chloroplast ultrastructure and membrane lipidcomposition to the different degrees of frost resistance achieved in leaves of spinach,ivy, and spruce. J. Plant Physiol. 117, 41–55.SUNG, D.-Y., KAPLAN, F., LEE, K.-J., GUY, C. L., 2003: Acquired tolerance to temperatureextremes. Trends Plant Sci. 8, 179–187.THOMAS, R. J., GRETHLEIN, A. J., PEROU, C. M., SCHEIRER, D. C., 1990: Translocation inPolytrichum commune (Bryophyta). III. Loading of sugars in source leaves. Amer. J.Bot. 77, 1574–1581.THOMAS, R. J., RYDER, S. H., GARDNER, M. I., SHEETZ, J. P., NICHIPOR, S. D., 1996:Photosynthetic function of leaf lamellae in Polytrichum commune. Bryologist 99, 6–11.THOMASHOW, M. F., 2001: So what’s new in the field of plant cold acclimation? Lots! PlantPhysiol.125, 89–93.UEMURA, M., STEPONKUS, P. L., 1997: Effect of cold acclimation on the lipid compositionof the inner and outer membrane of the chloroplast envelope isolatedfrom rye leaves.Plant Physiol. 114, 1493–1500.WRISCHER, M., LJUBE[I], N., SALOPEK, B., 1998: The role of carotenoids in the structuraland functinal stability of thylakoids in plastids of dark-grown spruce seedlings. J. PlantPhysiol.153, 46–52.WRISCHER, M., LJUBE[I], N., MAGNUS, V., DEVIDÉ, Z., 2000: Structural and functionalcharacteristics of overwintering blackberry leaves. Acta Bot. Croat. 59, 5–16.226 ACTA BOT. CROAT. 64 (2), 2005LJUBE[I] N., WRISCHER M., PREBEG T., DEVIDÉ Z.

Structural changes of lamellar cells in leaves of the moss Polytrichum formosum Hedw. during winter freezing and thawing processes

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