Ultrastructure and Lipid Composition of Mosses

Some moss gametophores, after fixation for electron microscopy, show strong accumulation of osmium in cytoplasmic droplets. The droplets are numerous in gametophores of Ceratodon purpureus (Hedw.) Brid., Tortula ruralis (Hedw.) Gaertn., Mayer & Scherb. and Hypnum curvifolium Hedw.; there are fewer in Mnium cuspidatum Hedw.; and there are none in Mnium punctatum Hedw. var. elatum Schimp. and Hygrohypnum luridum (Hedw.) Jenn. Lipid analyses of these gametophores showed a correlation between the amount of triglycerides with the occurrence of cytoplasmic droplets. The same correlation was encountered within one species. Cultures of C. purpureus had numerous cytoplasmic droplets when the triglyceride content was high, but had few when the triglyceride level was low. of mosses contain, besides fatty acids common for plant lipids, acids with 4 and 5 double bonds, such as eicosatetraenoic (arachidonic, abbreviated 20:4) and eicosapentaenoic (20:5) acids (Gellerman et al., 1972; Anderson et al., 1974). Some species also contain acetylenic fatty acids, mainly 9,12,15-octadecatrien-6-ynoic (= 18:3), accompanied in some cases by lesser amounts of 9,12-octadecadien-6-ynoic (= 18:2) and 11,14-eicosadien-8-ynoic (= 20:2) acids (Andersson et al., 1974; Anderson et al., 1975). None of these occur in seed plants where acids with 2 and 3 double bonds, octadecadienoic (linoleic, 18:2) and octadecatrienoic (linolenic, 18:3) usually are those of highest unsaturation. Osmium tetroxide reacts with double bonds to form esters of osmic acid (Criegee, 1936; Korn, 1967). This heavy metal is used in tissue fixation for electron microscopy, wherein electron dense areas are often interpreted as representing the cellular sites of unsaturated lipids. Some mosses show cytoplasmic droplets of unusually high electron density after fixation with OsO4. These spherical bodies have been reported in the cytoplasm of gametophores from Tortula ruralis (Tucker et al., 1975), Ceratodon purpureus (Valanne, 1971a), Polytrichum juniperinum (Bonnot & H6bant, 1970), Mnium spp. (Eyme & Suire, 1967) and others. Such droplets have also been observed in sporogenous tissue of Mnium spp. (Eyme & Suire, 1971) and in spores of Polytrichum commune (Karunen, 1This investigation was supported in part by U.S. Public Health Service Grant AM 05165 from the National Institutes of Health; U.S. Public Health Service Grant HL 08214 from the Program Project Branch, Extramural Programs, National Heart Institute; and by the Hormel Foundation. We thank Dr. W. P. Cunningham for helpful discussions and use of EM facilities, and Dr. J. D. Bewley for moss samples. 2 Present address: Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108. 8 The Hormel Institute, University of Minnesota, Austin, Minnesota 55912. This content downloaded from 157.55.39.203 on Thu, 20 Oct 2016 04:16:50 UTC All use subject to http://about.jstor.org/terms 340 THE BRYOLOGIST [Volume 79 1972). Some authors have considered them to be lipid droplets since they are similar in size, shape and location to the structures which contain stored lipids in the cells of animals and higher plants (Fawcett, 1966; Frey-Wyssling & Miihlethaler, 1965). The purpose of this study is to test this assumption by comparing lipid composition with the ultrastructural observations. The results from several field collections of mosses and from one species grown under different conditions verify the concept. MATERIALS AND METHODS Mosses.-Ceratodon purpureus (Hedw.) Brid. was collected from sand in an exposed, shallow ditch; Mnium punctatum Hedw. var. elatum Schimp, from a moist, shaded depression; Hypnum curvifolium Hedw. from a dry, exposed plank walkway near the edge of a bog. All of these stands were in the Cedar Creek Natural History Area (University of Minnesota), Anoka County, Minnesota. Mnium cuspidatum Hedw. was collected from a partially shaded roadside verge, J. C. Hormel Nature Center, Austin, Minnesota; Tortula ruralis (Hedw.) Gaertn., Mayer and Scherb. from banks of the Bow River in deep shade of spruce trees, Calgary, Alberta, Canada. The latter sample was received in the dry state and was rehydrated in the fixative for electron microscopy, and in water for 0.5 hr. to facilitate lipid extraction. Hygrohypnum luridum (Hedw.) Jenn. was collected from rocks in a small stream near Exshaw, Alberta, Canada. The sample was obtained in moist condition and was maintained in 5 gal. glass tanks containing 100 ml inorganic medium diluted with distilled water to 1.5 liter. The water level was maintained and nutrients periodically replenished. The moss was grown under a 14 hr. light cycle, 300 f.c. fluorescent light at 25' for 3 months before electron microscopy and lipid analysis. Axenic liquid cultures of Ceratodon purpureus protonemata were established using spores released from capsules which had been surface sterilized for 5 min. in 2.5% chlorine bleach. Four to six capsules were used to inoculate each of several 4 oz. prescription bottles which contained 10 ml of medium. The inorganic medium was as described by Mueller (1973) and all cultures were grown at 250 f.c. fluorescent and incandescent light, 18 hr./day at 18-20'. After 2 weeks protonemata from four 4 oz. bottles were combined by pouring into a 16 oz. prescription bottle containing 25 ml fresh medium. Fourteen days later all the growth medium was removed from each culture by means of a sintered glass filter stick and then 20 ml fresh medium was added to each culture. The experimental period was 8 weeks during which the nutrients of some cultures were replenished 4 times at intervals of 11 days, by addition of 20 ml of growth medium. Other cultures were stressed by not adding nutrients. All cultures were harvested at the end of 8 weeks for electron microscopy and lipid analysis. Electron microscopy.-Leaves of the different moss species were sliced into 1 mm pieces in fixative and protonematal filaments placed intact in fixative according to Karnovsky (1965), modified to a concentration of 4% formaldehyde and 5% glutaraldehyde in 0.1 M phosphate buffer of pH 7.0. After 4-12 hr. of aldehyde fixation the tissue was post-fixed for 12-16 hr. in 1-1.5% OsO4 solution in the phosphate buffer. It was then dehydrated in 5 successive steps by treatment with acetone-water, increasing the concentration from 30 to 100% acetone. The tissue was embedded in Spurr's epoxy resin. After staining in aqueous uranyl acetate followed by lead citrate, the sections were viewed with a Hitachi HU-11C electron microscope. Analysis of lipids.-Lipids were extracted from hydrated gametophores or from protonemata and analyzed according to Gellerman et al. (1972, 1975). This involved extraction of the materials by mixtures of chloroform and methanol in an Omnimixer. After recovery of the crude lipids, they were separated by thin layer chromatography. Lipid classes were identified by appropriate spray reagents and by Rf values in reference to authentic compounds. Their amounts were assayed visually by thin layer chromatography taking reference to chromatograms of the known compounds in appropriate amounts. Fatty acid compositions were obtained by acidic methanolysis of the lipids and subsequent gas liquid chromatography of the fatty acid methyl esters. RESULTS AND DISCUSSION Micrographs of mosses from their natural habitat show osmiophilic spherical bodies, as they have been reported by others (see above), ranging in size from 0.15 to 0.25 um in diameter (Fig. 1-3). They are numerous in the cytoplasm of Ceratodon purpureus, This content downloaded from 157.55.39.203 on Thu, 20 Oct 2016 04:16:50 UTC All use subject to http://about.jstor.org/terms TABLE 1. Cytoplasmic droplets and lipid classes' of moss gametophores. Wax + Free fatty Total steryl acids + GlycoPhosphoDroplets Lipids Triglycerides esters diglycerides lipids lipids Ceratodon purpureus Many 12.4 66 18 traces 7 8 Tortula ruralis Many 8.4 37 8 traces 36 19 Hypnum curvifolium Many 9.5 49 6 2 30 13 Mnium cuspidatum Few 12.7 28 3 traces 40 29 Mnium punctatum None 11.9 3 13 13 47 23 Hygrohypnum luridum None 8.0 2 5 1 73 19 ' Percent of total lipids. 2 mg lipids obtained/100 mg extracted and dried residue. ?,D