Observations of fluorescence with ultraviolet microscopy and staining with transmission electron microscopy indicate that a substance reacting like callose is present as a plugging material in pitlike areas in the walls of mature cork (phellem) cells

Krahmer, Robert L.

Litvay, John D.

English

Wood and Fiber Science (E-Journal)

THE PRESENCE OF CALLOSE IN CORK CELLS' John D. Litvay and Robert L. Krahnzer Assistant P~.ofessor, School of Forest Resources, University of Maine, Orono, ME 04474 ant1 Professor of Forest Products, School of Forestry, Oregon State University, Corvallis, OR 97331 (Received 13 hlay 1976) ABSTRACT 0l)servations of fl~~orescence wit11 ultraviolet nlicroscopy and staining with transmission electron microscopy indicate that a substance reacting like callose is present as a plugging material in pitlike areas in the walls of mature cork (phe l l en~)  cclls. Keytoorrls: Pseudotstga merrziesii, Abies spp., Tsuga heterophylla, Acer mucro)~hylltlm, <)uercus suher, cork, callose, phellenl, 111traviolet microscopy, transmission clectron ~nicros- copy. INTRODUCTION Callose is a carbohydrate, which is com- posed of glucose units linked P-1,3 (Esch- rich 1956; Fu ct al. 1972), and is present in the walls of sieve and epidermal cells of higher plants ( Fahn 1974). Although its fullction in these cells is unknown, it is thought to act primarily as a plugging ma- terial ( Currier 1957). Currier and Strug- ger ( 1956) suggest that callose might play an even greater role than this in cells and coultl be distributed more widely in plants than is suspected now. In phloem tissue of bark, callose can be seen as a plugging ~naterial in the sieve areas of the sieve cells. Sitte (1955) studied the cork cell wall of cork oak (Quercus suljer L.) and observed in the pits a plug- ging material that he was ullable to iden- tify, but referred to as being "alien to su- berill." We have observed similar plugging of pitlike areas in the walls of mature phel- lem (cork) cells in Douglas-fir [Pseudo- tsugu menzie~ii ( Mirb. ) Franco] bark ( Fig. l ) ,  although to our knowledge the presence of callose in cork cells has not been re- ported previously. The presence of callose in situ usually is determined by the fluorescence observed 1,ecause of ultraviolet ( U V )  light excitation ' Thc project was supported in part by hlcIntire! Stenllis funds. Paper 1084, Forest Research Laho- ratory, School of Forestry, Oregon Stat(. University. when the callose is stained with resorcin blue or aniline blue (Currier 1957; Esch-- rich and Currier 1964). Recause phenolics in lignin and extractives fluoresce, they can mask any fluorescence produced by callose if they are present in the same cells. There- fore, use of the fluorescence phcnomenolr for determining the presence of callose is applicable only where these phenolics are not present or can be removed from the cell without removing the callose. Also, thc ability to see callose with the tr;~nsmission electron microscope (TEM) has been hin- dered by the fact that electron microscopy stains do not differentiate cellulose from callose ( Esclirich 1956). Because of these limitations, callose may have been over- looked in many cells. On the basis of re- sults of procedures described below to over- come these limitations for observing callose in these cells, we believe there is substan- tial evidence for the presence of callose in the phellem cells of Douglas-fir, as well as in true firs (Abies spp.), western hemlock [Tsuga heteroplzylla ( Kaf. ) Sarg.], bigleaf maple ( Acer rr~uc~oph yllum Pursh ) , and cork oak. MATERIALS AND METHODS Tissue collection Douglas-fir bark was collected from freshly felled logs and immediately cross- cut into 'h-inch slabs from which cork layers were excised with a razor blade. \\ 001) A N D  FIRER 146 FALL 1976, V. 8 ( 3 )  CALLOSE IN CORK CELLS 147 148 JOHN D. LITVAY AND ROBERT L. KKAHhIEH FIG. 7. Fluorescence in cork cells after saponi- fication for 30 inin with 3% aqueous NaOH and then stained with aniline blue. The fluorescing pegs (arrows) result from staining with aniline blue and indicate the presence of callose, hut the fluorescence in the cell wall is the same as that seen in Fig. 3. FIG. 8. Scanning electron micrograph of 4 pegs protruding upward from the compound middle lamella of cork, saponified for 30 min with warn1 370 aqueous NaOH. FIG. 9. Longitudinal section through a callose peg (arrow), attached to the compound middle lamella. The black material lining the peg and wall is the metal coating used on these cork cells for examination with SEM before sectioning for TEhl (section stained with 1% osmium). F~c .  1 .  Section through adjacent cork cell walls from Douglas-fir bark, which shows layering in thc w;ills. A plugged pitlike area extends through zone 1 (co~npound middle lamella) and zone 2 of the cell on the right. The section was pretreated with periodate ( P )  and thiosemicarhazide (TSC) ,  and stai~led with 1% osnlicini. Frc. 2. Fluorescence in transverse section of unextrncted and unstainetl cork cells. The fluorescence is c;u~sed by phenolics present in the cell walls. F'I~:. 3. Reduction of fluorescences in cork cells after ~a~onificatio11 with warm 3% aqueous NaOH for SO rnin (unstained transverse section). Frc. 4. Section of adjacent cork cell walls that have been extracted with benzene-ethanol (section pretreated with P and TSC; stained with 1% osiniuni). F'ic. 5. Cork cell walls extracted with benzene-ethanol ;ind amn~onium oxilate. The two large white tireas in the compound il~iddle lamella are unstained substances plugging these areas (section pretreated with P and TSC; stained with 1% osmiun~). FIG, 6. Cork cell walls saponified with warill 3% aqueous NaOH for 30 min. Only the compound nliddlc larllella remains (pretreated with P and TSC; stained with 1% osininm). CALLOSE IN CORK CELLS 149 (,'hemica1 treatments ant1 staining thiosemicarbazide for 1% h at room temper- Solvent extractioils and selected staining of cork were done on 24-pm-thick sections. The treated sections then were prepared for optical and electron microscope observa- tions. Although Douglas-fir bark was the priilcipal species studied, barks from true firs, western hemlock, bigleaf maple, and cork oak were treated and stained similarly to that developed for Douglas-fir. Solvent extraction. The extraction was as follows: 1. Benzene-ethanol extraction ( 1: 1 v/v, 24 h at rcflux) ; 2. Absolute ethanol extraction (24 h at reflux) ; 3. Hot water extraction (24 h at reflux); 4. Ammonium oxalate estractioil (0.5% solution, 24 h at reflux) and; 5. Saponification with warm 3% aqueous NaOH ( 30 min ) . Staining ant1 microscop!/. Unstained sec- tiol~s were mounted in glycerine for obser- vations of fluorescence with ultraviolet light. To cause callose to fluoresce, sections were stained with a 1% aqueous solutioil of aniline blue ( p H  not adjusted) for 30 mi11 (Currier and Strugger 1956). A Zeiss U~liversal microscope with Zeiss UV at- tachments was used (Exc:ter filter BG :%; I~arrier filter 50). C:ork prepared for scanni~~g electron mi- croscopy ( SEM ) was removed from the ex- traction sequence, solvent exchanged into trifluoromethane, and critical point dried. Surfaces were coated with gold/paladium (60:40) and observed with an I.S.I. mini- scamling electron microscope. To differentiate cellulose from callose in tra~ismission electron microscopy, the cellu- lost, was stained with 1% aqueous osmium, using a pretreatment procedure described by Seligman et al. ( 1965). The pretreat- ment was: soaking 20-pm-thick sections in a freshly made solution of 1% sodium per- iodate at room temperature for 3 h, rinsing the sections in distilled water, and then soaking them in a fresh aqueous solution of acetic acid (25%,), which contained 1% ature. After this treatment, the sections were washed with distilled water and stained with 1% aqueous solution of osmium tetroxide at room temperature for 3 h. Stained samples were embedded in Spurr's media (Spurr 1969), sectioned with a dia- mond knife, and examined in a Phillips Ehi- 300 transmission electron microscope. RESULTS AND DISCUSSION UV microscopy and fluorescence Untreated arid unstained sections of cork exhibited green-yellow fluorescence in the cell wall when observed with the UV micro- scope (Fig. 2) .  This fluorescence still was present in the cork cells after cork sections had been extracted chemically through the ammonium oxalate step of the extraction sequence. After saponification, however, the amount of inaterial in the cork sections that fluoresced was reduced (Fig. 3) ,  and the color emitted was pale yellow. Chemi- cal analysis of the solubles from the saponi- fication indicated that they were phenolic, and examination of these phenolics under UV light showed that they exhibited a greenish-yellow fluorescence. Use of TEM showed that zones 3 and 4 (Fig. 1) had been removed from phellem cells that were extracted with benzene- ethanol (Fig. 4) .  No further wall layers were removed with hot water and ammoni- um oxalate extractions (Fig. 5 ) .  After saponification, however, only the corn- pound middle lamella remained (Fig. 6 ) .  The reduction of fluoresceilce after saponi- fication (Fig. 3 )  could be attributed to the removal of zone 2 in the cork cell wall, which is a suberin layer with considerable a mer phenolics present (Litvay and Kr. h 1977). After the suberin layer was removed, callose could be detected when the callose was stained to fluoresce. Normally, alkaline coilditions are used for the staining, which makes callose fluoresce a bright yellow. When we did not adjust the pH of the aque- ous solution of aniline blue stain, however, the fluorescence given off by the callose was yellow-green, which made it inore dis- cernible from the compound middle lamella, which fluoresces yellow. I11 certain loca- tions, the appearance of yellow-green fluo- rescing "pegs" could be seen easily, which indicated the presence of a calloselike ma- terial. Figl~re 7 is of cork, saponified for 30 n1in and stained with aniline blue. The diaiileters of the fluorescing pegs range f ron~  0.2 to 0.3 pm The distribution of fluo- rescing pegs is variable, but they were seen in all sections and growth increments of inatllre Douglas-fir cork studied. To observe these pegs with electron mi- croscopy, the same cork sections that were stailled and used for UV observatiolls were critical point dried, mctal coated, and ob- served i11 an SEM. Figure 8 is a scanning electron micrograph froin the same cork section used in Fig. 7 and shows four of these pegs. After the cork sections had been observed with SEM, thcy were re- moved from the specimen stubs and stained with a 1% aqueous solution of osmium tetroxide for 3 h at room temperature. They then were embedded in Spurr's media (Spurr 1969), sectioned, and observed in a TER4. Figure 9 is a loi~gitudinal section of a peg attached to the compound middle lamella (zone 1) of the cell. Trut~srnission electron rrticroscopy and staining Pretreating cork sections with sodium periodate and thiosemicarbazide before staii~ing them with osnlium tetroxide dif- ferentiated callose fro1-n cellulose in ultra- thin sections observed with TEM. In the pretreatment reaction, periodate cleaves ad- jacent hydroxyls and simultaneonsly oxi- dizes the cleaved hydroxyls to aldehydes. The thiosernicar1):izide then condenses with the aldehydes, which leaves an aldehyde- thiosemicarbazone that will readily stain wit11 osmium tetroxide (Seligman et al. 1965). For this reaction to occur, the car- bohydrate being oxidizccl must have ad- jacent hydroxyls. Cellulose is linked P-1,4 and has adjacent hydroxyls at Carboils 2 and 3, but callose is linked 8-1,3 and does not possess adjacent hydroxyls. Thus, cellu- lose will react and stain with osmium, but callose will not. In areas of a cell wall that contain callose surrounded by cellulose, the callose appears white (unstained), sur- rounded by dark stained cellulose. Untreated and pretreated cork, stained with osmium tetroxide, were examined with TEM. Figures 1, 4, 5, and 6, which have already been used to show the ren~oval of wall layers with various solvents, illustrate results of the pretreatment and stainiag pro- cedure for observation of callose. The effect of the pretreatment is seen in the compouild middle lamella (zone 1:). The zone stains darker l~ecause of the pre- treatment, which indicates that oxidizable carbohydrates are present in this zone. Without the pretreatment, the compound middle lamella is about as light as zone 3 labeled on Fig. 1. I11 neither Fig. 1 nor Fig. 4 are there white regions, surrounded by dark stained material, to indicate the pres- ence of callosc,. After sections extracted with benzene-ethanol, however, have bee11 extracted further with hot water or arn- monilim oxalate, white areas appear in the compound middle lamella (Fig. 5 ) .  The diameters of these areas are comparable to the diameters of the fluorescing pegs shown in the UV study. Although these white areas appear in the inicrograph as empty holes in the compound middle lamella, 110 such holes or gaps are seen in sections ex- tracted with the same solvents, but not pre- treated before staining. SEM examinations did not show holes in any of this extracted or saponified material. Because white areas do not appear in the conlpound middle lamella until after extrac- tion with hot water or ammoniurn oxalate, other material, such as pectins or hemi- celluloses, which are soluble in these sol- vents, appear to be associated with the cal- lose plugs and must be removed to prevent them fro111 masking the callose. This is partially substantiated by Fu et al. (1972). To isolate callose, they had to treat the car- bohydrate with a-amy1a::e and extract it with hot water to reinove associated starch and hemicelluloses. The UV study showed that thc callose CALLOSE I N  CORK CELLS 151 pegs extended into the suberin layer (zone 2 ) ,  although Fig. 5 does not show white areas present in that layer. Again, the cal- lose in the suberin layer could be associ- ated with water insoluble materials similar in composition to those found in the suberin layer, such as phenolics, which stain with oslniuin and would mask the presence of callose. In the colnpound middle lamella, holvcver, the materials reacted to this pre- treatment arlcl staining in a manner that would suggest the presence of callose in the pitlike areas. CONCLUSIONS 1) A lnaterial that gives a callose rc- action to aniline blue under UV light is prc,scnt in mature saponified cork (phel- lenl) cells of Ilouglas-fir bark. Similarly, a material is present in the compound mid- dlc lan~ella of cork cells extracted with or- ganic solvents and hot water or ammonium oxalate, tvhic11 is not stained with osmium tetroxide after treatment with periodate and thiosemicarbazide. Both of these re- sults provide substantial evidence for the presence of a calloselike substance. Ob- scrvations on mature cork from the true fir\, western hemlock, bigleaf maple, and c o ~ k  oak yielded similar results. 2 )  Callose in plant cells functioils as a plugging material, and in cork cells it is plugging pitlike areas in the cell wall. REFERENCES CURHIEH, H. B. 1957. Callose substance in plant cells. Am. J. Bot. 44:478-488. CURHIER, H. B., ASD S. STHUGCER. 1956. Ani- line blue and fluorescence microscopy of c:al- lose in bnlh scales of Allitcm cepu L. Proto- plasm 45: 552-559. ESCHRICEI, W. 1956. Kallose (Ein kritischcr Sammclhericht ) . Protoplasm 47:587-530. ESCHRICH, W., AND H. 13. CURRIER. 1664. Iden- tification of callose by its diachronle and fluorescence reactions. Stain Tech. 39:303- 307. FAHN, A. 1974. Plant anatomy. Oxford, Per- gainon Press Ltd. Fu, Y. I., P. J. GUTMANN, AND T. E. TIMELL. 1972. Polysaccl~arides in the secondary phloem of Scots pine (Pinus sylvestris L.) I. Isolation and characterization of callose. Cellulose Chem. Technol. 6:507-512. LITVAY, J. D. AND R. L. KHAHAIEH. 1977. Wall layering in Douglas-fir cork cells. Wood Sci- ence 9(  4 ) : 167-173. SELIGTIIAN, A. hf., J. S. HANKER, H. WASSERKRUG, H. Dnxoc~ows~r ,  AND L. KATZOFF. 1965. Histochemical demonstration of some oxi- dized i~~acromolecules with thiocarbohydrazide ( TCH) or thiosemicarbazide ( TSC) and os- mium tetroxicle. J. Histochem. Cytol. Chem. 13:629-637. SITTE, P. 1955. I>er feinbau verkorkter zell- wande. Mikroskopie 10: 178-200. SPUHH, A. T. 1969. A low viscosity epoxy resin enlhedding medium for electron microscopy. J. Ultrastruct. Res. 20:31-43. 

The Presence of Callose in Cork Cells

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The Presence of Callose in Cork Cells

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