Comparison of the ultrastructure of conventionally fixed ... - Springer Link

Protoplasma (1990) 157:64-74

OTON.ASMA 9 by Springer-Verlag 1990

Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips of Nicotiana and Arabidopsis J. Z. Kiss l' *, Th. H. Giddings Jr. 2, L. A. Staehelin 2, and F. D. Sack 1 Department of Plant Biology, Ohio State University, Columbus, Ohio, and 2Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado Received December 26, 1989 Accepted February 23, 1990 Dedicated to the memory of Professor Oswald Kiermayer

Summary. To circumvent the limitations of chemical fixation (CF) and to gain more reliable structural information about higher plant tissues, we have cryofixed root tips of Nicotiana and Arabidopsis by high pressure freezing (HPF). Whereas other freezing techniques preserve tissue to a relatively shallow depth, HPF in conjunction with freeze substitution (FS) resulted in excellent preservation of entire root tips. Compared to CF, in tissue prepared by HPF/FS: (1) the plasmalemma and all internal membranes were much smoother and often coated on the cytoplasmic side by a thin layer of stained material, (2) the plasmalemma was appressed to the cell wall, (3) organelle profiles were rounder, (4) the cytoplasmic, mitochondrial, and amyloplast matrices were denser, (5) vacuoles contained electron dense material, (6) microtubules appeared to be more nmnerous and straighter, with crossbridges observed between them, (7) cisternae of endoplasmic reticulum (ER) were wider and filled with material, (8) Golgi intercisternal elements were more clearly resolved and were observed between both Golgi vesicles and cisternae, and (9) iarger vesicles were associated with Golgi stacks. This study demonstrates that HPF/FS can be used to successfully preserve the ultrastructure of relatively large plant tissues without the use of intracellular cryoprotectants.

Keywords: Cryofixation; Tobacco; Uttrarapid freezing. Abbreviations:CF chemical fixation; ER endoplasmic reticulum; FF freeze fracture; FS freeze substitution; ItPF high pressure freezing. Introduction Plant r o o t tips are c o m p o s e d o f m a n y different types o f cells engaged to varying degrees in division, growth, and secretion. Electron microscopy of chemically fixed * Correspondence and reprints, present address: Department of Moiecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309-0347, U.S.A.

root tips has led to i m p o r t a n t insights into the relationship between structure and function o f these cells. However, due to the slow rate o f chemical fixation (Mersey and McCully 1978, Gilkey and Staehelin 1986), precise structural information on dynamic cellular events has remained rather sketchy, thereby limiting in critical ways o u r understanding o f the functional aspects o f m a j o r cellular processes. M o s t of the limitations of chemical fixatives can be overcome by the use o f cryofixation techniques, providing the sample can be frozen without the f o r m a t i o n o f d a m a g i n g ice crystals (reviewed in Gilkey and Staehelin 1986). U l t r a r a p i d freezing under atmospheric pressure can preserve samples without ice crystal form a t i o n to a depth of a p p r o x i m a t e l y 10 g m when cooled f r o m one surface (e.g,, cold block freezing) and to an overall m a x i m u m thickness o f a b o u t 40 g m when cooling is applied f r o m both sides ( p r o p a n e jet freezing). Despite these severe limitations, studies o f such cryofixed specimens have yielded exciting new i n f o r m a t i o n on a variety o f animal (Heuser et al. 1979, Bridgman and Dailey 1989) and plant tissues (Fernandez and Staehelin 1985, Guinel and McCuUy 1987), as well as a variety o f smaller specimens such as t r y p a n o s o m a t i d s (Linder and Staehelin 1979), unicellular algae (Giddings et al. 1980, Nicolas et al. 1987), pollen tubes (Lancelle et al. 1987, Tiwari and Polito 1988), stamen hairs (Lancelle et al. 1986), fungal hyphae ( H o w a r d and Aist 1979, H e a t h and K a m i n s k y j 1989), and plant suspension culture cells (Staehelin and C h a p m a n 1987).

J. z. Kiss et al.: Comparison of CF and HPF/FS of root tips To freeze biological samples thicker t h a n 40 g m w i t h o u t the f o r m a t i o n o f d a m a g i n g ice crystals, the samples have to be either p r e t r e a t e d with a chemical c r y o p r e servative such as glycerol, which m a y i n t r o d u c e artifacts, o r frozen u n d e r high pressure. U n d e r o p t i m a l c o n d i t i o n s , high pressure freezing ( H P F ) at 2100 b a r can p r o d u c e excellent freezing o f biological samples u p to 600 g m in thickness ( M o o r 1987, C r a i g a n d Staehelin 1988, D a h l a n d Staehelin 1989). Since a c o m m e r c i a l H P F a p p a r a t u s has only recently b e c o m e available, the n u m b e r o f systematic s t r u c t u r a l studies o f b i o l o g i c a l samples utilizing H P F is still r a t h e r limited, a n d basic questions c o n c e r n i n g specimen p r e p a r a t i o n a n d p r o cessing, high pressure freezing artifacts, a n d interpret a t i o n o f images still need to be addressed. N e v e r t h e less, a variety o f high pressure frozen cells a n d tissues have been successfully e m p l o y e d for freeze fracture ( F F ) a n d freeze s u b s t i t u t i o n (FS) studies. These include investigations o f cartilage tissues ( F F , FS: H u n z i k e r et al. 1984), p i t u i t a r y cells ( F F , FS: D r a z n i n et al. 1988), r o o t tips ( F F : C r a i g a n d Staehelin 1988; FS: K a e s e r et al. 1989), rust fungi ( F F : K n a u f a n d M e n d g e n 1988), p o l l e n tubes (FS: Lancelle a n d H e p l e r 1989), a n d p l a n t leaves (FS: W e l t e r et al. 1988). H o w e v e r , in this latter study, structural p r e s e r v a t i o n m a y have been affected by the use o f m e t h a n o l as a c r y o p r o t e c t a n t . H e r e we r e p o r t o n the u l t r a s t r u c t u r a l a p p e a r a n c e o f high pressure frozen/freeze s u b s t i t u t e d cells, frozen w i t h o u t the use o f i n t r a c e l l u l a r c r y o p r o t e c t a n t s .

65 or less. To test whether dextran alters structure, root tips were placed in the dextran solution for 30 rain and then examined by light microscopy. Root tip cells had a normal appearance and were not plasmolyzed.

Freeze substitution Specimen cups were transferred at liquid nitrogen temperature to 1.8ml cryogenic vials (Vangard International, Neptune, N.J., U.S.A.) filled with 1% (w/v) osmium tetroxide in acetone as a substitution medium. Substitution was carried out at - 78.6 ~ in a dry ice/acetone bath for 2.5 d followed by 2 h at - 20 ~ 2 h at 0 ~ and 2 h at 23 ~ The samples were then rinsed in acetone, infiltrated and embedded in Spurr's resin. In some cases, 0.1% (w/v) uranyl acetate was added to the substitution medium, and the material was processed as described above. In another protocol, substitution was performed with 1% (w/v) osmium tetroxide in methanol for 1.5 d, and processing was as above.

Chemicalfixation and electron microscopy Chemical fixation and subsequent processing were essentially as described by Sack and Kiss (1989), except that postfixation was for 2 h. Briefly, seedlings were fixed with 1% (w/v) paraformaldehyde and 2% (v/v) glutaraldehyde, and postfixed in 2% (w/v) osmium tetroxide. Sections of chemically fixed samples were stained with aqueous uranyl acetate and lead citrate (Reynolds 1963). To improve the contrast of freeze-substituted samples, sections were stained with 2% (w/v) uranyl acetate in methanol, rinsed in a graded methanol series, and then stained with lead citrate. Sections were observed at 60-80 kV in either a Zeiss 10 or a Philips CM 10 electron microscope.

Results Evaluation of freezing and substitution media and identification o f H P F artifacts

Materials and methods Plant material and culture conditions Roots of Nicotiana sylvestris Speg. et Comes (a diploid species of tobacco; Kiss and Sack 1989) and of Arabidopsis thaliana (L.) Heynh. (Columbia) were examined. Culture conditions were essentialiy as described in Kiss et al. (1989). Briefly, seeds were surface sterilized, and seedlings were grown under sterile conditions in vertically-oriented Petri dishes on 1% (w/v) agar containing nutrients and 1% (w/v) sucrose under continuous illumination at approximately 23 ~ Roots were used when 4-9 mm long, i.e., 6-7 d after sowing (Nicotiana) or 2.5 d after sowing (Arabidopsis).

High pressurefreezing The tip most 1-2ram of the root was excised with a razor blade. Three to four root tips were placed in each specimen cup (designed by Craig et al. 1987) which was coated with a freshly prepared vegetable lecithin solution (100mg/ml in chloroform). The specimen cups were filled with either distilled water, 1-hexadeeene (an inert paraffin oil), or 15% (w/v) aqueous dextran (MW 38,800). Specimens were then frozen in a Balzers HPM 010 apparatus as described by Craig and Staehelin (1988) and were stored in liquid nitrogen until freeze substitution. The time between excision of the root tips and freezing was 5 rain

The best s t r u c t u r a l p r e s e r v a t i o n was o b t a i n e d b y freezing r o o t tips i m m e r s e d in 15% a q u e o u s d e x t r a n (Figs. 1-3). This m e t h o d p e r m i t t e d successful H P F o f entire r o o t tips o f b o t h Arabidopsis a n d Nicotiana. T h e d i a m e t e r o f the r o o t a b o v e the r o o t cap was a p p r o x i m a t e l y 1001am for Arabidopsis a n d a p p r o x i m a t e l y 130 g m for Nicotiana. N o ice crystal d a m a g e was visible at the electron m i c r o s c o p e level either in nascent cells o f the stele in the center o f the r o o t (Fig. 1) o r t h r o u g h o u t the r o o t c a p (Fig. 2). M e m b r a n e s such as the end o p l a s m i c r e t i c u l u m (ER), the p l a s m a m e m b r a n e , the vacuole m e m b r a n e (tonoplast), a n d the n u c l e a r envelope were s m o o t h , a n d organelles a p p e a r e d t u r g i d (Fig. 3). The nuclear m a t r i x , which is p a r t i c u l a r l y susceptible to ice d a m a g e , exhibited no signs o f ice crystal f o r m a t i o n (Fig. 3). O n e artifact seen even in otherwise well-preserved m a t e r i a l was b r o k e n cell walls. These were seen m o s t frequently n e a r the r o o t cap p e r i p h e r y a n d o c c a s i o n a l l y d e e p e r within the tissue (Fig. 4). In a few o f the p e r i p h e r a l cells o f Arabidopsis r o o t caps,

Figs. 1 and 2. Low magnification electron micrographs of roots prepared by HPE/FS. Roots were frozen in dextran. FS was with osmium tetroxide in acetone. Entire root tips are well-preserved without visible ice crystal damage. Bars: 10 gm Fig. 1. Nascent ceils of the stele from the center of a Nieotiana root. Note cell plate formation (arrowhead) Fig. 2. Arabidopsis root cap. C Columella cell; M mucilage; P peripheral cell

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Figs. 3-6. Effects of freezing media on the quality of preservation by HPF/FS. A Amyloptast; D dicytosome; M mitochondria; N nucleus Fig. 3. Central columella cell of a Nicotiana root that was frozen in dextran. Good preservation is indicated by a dense cytoplasmic and nucleoplasmic matrix (without ice crystal damage) and by smooth membrane profiles. Note vacuoles (V) filled with electron dense material. Bar: 5 gm Fig. 4. Peripheral cell of an Arabidopsis root cap frozen in dextran. Some of the large, mucilage containing-vesicles appear to have burst (arrowhead). Arrow indicates artifactual crack in ceil wall. Bar: 2 gm Fig. 5. Central columella cell of a Nieotiana root frozen in hexadecene. Cracks (arrowheads) appear throughout the cytoplasm and in cell walls, although localized areas are well-preserved (asterisk). Bar: 5 gtm Fig. 6, Meristematic cell of an Arabidopsis root frozen in distilled water. Ice-crystal damage is present throughout the cell. Bar: 5 gm

s o m e o f the large, m u c i l a g e - c o n t a i n i n g vesicles app e a r e d to h a v e b u r s t , p o s s i b l y as a c o n s e q u e n c e o f the b r i e f e x p o s u r e o f the tissue to h i g h p r e s s u r e (Fig. 4). A t t e m p t s to use freezing m e d i a o t h e r t h a n d e x t r a n were

n o t as successful. T h e use o f h e x a d e c e n e as a freezing m e d i u m resulted in m o r e c r a c k s i n the c y t o p l a s m a n d in b r o k e n cell walls, a l t h o u g h t h e c y t o p l a s m was wellp r e s e r v e d in s o m e a r e a s (Fig. 5). I n s a m p l e s f r o z e n i n

68 distilled water, ice crystal damage was widespread throughout the root tip (Fig. 6). Material freeze-substituted simultaneously with osmium tetroxide and uranyl acetate in acetone was more difficult to section, perhaps because the uranyl acetate complexed with the dextran used in freezing. In this study, tissue that was substituted in methanol with osmium tetroxide appeared very extracted (data not shown). Freeze substitution in acetone with osmium tetroxide yielded the best results, and was the method used to generate most of the results reported here.

Comparison of ultrastruetural preservation with HPF/FS versus CF The ultrastructure of chemically fixed root tips (Figs. 7, 8, and 12) differed from that of high pressure frozen/ freeze substituted tissue (Figs. 9, 10, 11, and 13) in several significant aspects. Root cap columella cells prepared by HPF/FS had smooth membranes and turgid membrane-bound compartments (Figs.9, 10, 11, and 13) compared to chemically fixed samples which had wavy membranes and more collapsed membranebound compartments (Figs. 7, 8, and 12). With HPF/ FS, the plasma membrane was closely appressed to the cell wall (Figs. 9 and 10) while in CF, the plasma membrane was undulate and often separated from the wall (Figs. 7 and 8). In cells prepared by HPF/FS, the tonoplast was continuous, and vacuoles contained electron dense material (Fig. 9) whereas, in CF, the tonoplast was broken, and vacuoles appeared empty (Fig. 7). In HPF/FS, localized areas of the tonoplast had undulations in otherwise smooth regions (Figs. 3 and 9). The ground cytoplasm had a regular, evenly distributed matrix and appeared less extracted following HPF/FS (Fig. 9) compared to CF (Fig. 7). The ER cisternae were wider and filled with an electron dense material in high pressure frozen/freeze substituted material (Figs. 9 and t 1). The texture of the electron dense contents of the ER differed from that of

J.z. Kiss etal.: Comparisonof CF and HPF/FS of root tips the background cytoplasm (Fig. 11). In HPF/FS, some membranous elements (which may represent ER) of the developing cell plate (Fig. 1) stained more intensely than in CF (not shown). A thin, moderately stained coating on the entire cytoplasmic side of membranes was a general feature of high pressure frozen/freeze substituted cells. Such "coats" were observed on the plasma membrane (Figs. 9 and 10), mitochondria (Figs. 9 and 20), and Golgi vesicles (Figs. 9, 18, 19, and 22) as well as other membranes. Organelles h a d a distinctive appearance in high pressure frozen/freeze substituted material. Mitochondria were rounder and had a denser matrix (Fig. 9) than mitochondria from chemically fixed cells (Fig. 7). In HPF/FS, dictyosomes had prominent fenestrations and had large, spherical vesicles (Fig. 9). Amyloplasts were more spherical, and had a more uniform matrix with smoother thylakoids in HPF/FS (Fig. 13) than in chemically fixed samples (Fig. 12). Microtubules were intact, straight, and abundant in root tip cells prepared by HPF/FS (Fig. 14). In addition, HPF/FS preserved crossbridges between microtubules (Fig. 15). In chemically fixed material, microtubules were somewhat undulate and appeared to be less abundant (not shown), although this was not quantified. Only a relatively small amount of mucilage was visible on the exterior surface of the root cap following CF (not shown). In high pressure frozen/freeze substituted roots, thick layers of mucilage were preserved (Fig. 16). In the middle and outer regions of the mucilage, there were numerous electron translucent areas (absent in CF) that gave these regions a speckled appearance.

Golgi apparatus in columelIa and peripheral cells In chemically fixed Nicotiana root cap columella cells, dictyosomes had indistinct cisternae and vesicles, and intercisternal elements and fenestrations were difficult

Figs.7-11. Comparisonof preservation of Nicotiana columeltacell with HPF/FS vs. CF. CW Celt wall; M mitochondrias Fig. 7. Membranesare irregular and undulate, and the cytoplasmand vacuole (V) appear extracted.Dicytosome(D) cisternae are indistinct, and large Golgi vesicleswere not observed. Bar: 1gm Fig. 8. CF. High magnificationview of the plasma membrane (arrowheads)which is undulate. Bar: 0.05gm Fig. 9. HPF/FS. Membranesare smooth and regular, mitochondrial(M) profiles are round, and the cytoplasmicmatrix is dense.Vacuoles (lo are filledwith an electrondense matrix, and the tonoplast is intact (arrow) and generallysmooth but locallyundulate (asterisks). Large vesicles (arrowheads) are associatedwith dictyosomes(D). Bar: 1~tm Fig. 10. HPF/FS. High magnificationview of the plasma membrane(arrowhead) whichappears coated and closelyappressed to the cellwall (CW). Bar: 0.05gm Fig. 11. HPF/FS. The electrondensecontents(arrowhead)of the endoplasmicreticulum(ER)differin densityfromthe surroundingcytoplasm. Bar: 1gm

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J.Z. Kiss et al.: Comparison of CF and HPF/FS of root tips

Figs. 12 and 13. Amyloplasts (A) in Arabidopsis columella cells. Bars: 1gm Fig. 12. CF. The amyloplast envelope (arrow) is irregular and undulate Fig. 13. HPF/FS. Amyloplast profiles are round, and the envelope (arrow) is smooth. Thylakoids (arrowhead) are distinct and smooth Figs. 14-16. HPF/FS of Nicotiana roots. C W Cell wall Fig. 14. Cortical microtubules (arrowhead) in columella cells. Bar: 1gm Fig. 15. Crossbridges (arrowheads) between microtubules in a columella cell. Arrows indicate axes of microtubules. Bar: 0.1 gm Fig. 16. A thick layer of mucilage (/14)is present at the root cap periphery. In the middle and outer regions of the mucilage, there are numerous electron translucent areas that give these regions a speckled appearance. Bar: 1 gm

to discern (Fig. 17). Associated with dictyosomes were small vesicles that had a coat with the m o r p h o l o g y characteristic of clathrin (Fig. 17). F o l l o w i n g H P F / F S , a wide range of sizes of Golgi vesicles was observed, a n d large vesicles were associated with the trans face

(Fig. 18). Some vesicles were s u r r o u n d e d by a coat that did n o t have the m o r p h o l o g y characteristic of clathrin (Figs. 18 a n d 19). Intercisternal elements (Fig. 18) a n d fenestrations (Fig. 20) were observed in m a n y dictyosomes.

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Figs. 17-20. Dictyosomes in Nicotiana columella ceils, Bars: 0,5 gm (except for Fig, 19; Bar: 0,2 gm) Fig. 17, CF. Small, presumably clathrin-coated vesicles (arrowheads) are associated with the dictyosome Fig. 18. HPF/FS. Intercisternal elements (arrowheads) Fig. 19. HPF/FS. Larger Golgi vesicles are coated (arrowheads) Fig. 20. HPF/FS. Fenestrations (arrowheads) of dictyosome cisternae Figs. 21-23. Dictyosomes in Arabidopsis peripheral cells. Bars: 0.5 gm Fig. 21. CF. Golgi vesicles (V) contain a dense material with an uneven matrix Fig. 22. HPF/FS. Golgi vesicles (I0 are coated (arrows), and intercisternal elements (arrowhead) are prominent. Golgi vesicle contents are more uniform and more electron dense than in CF. Partially coated reticulum (asterisk) Fig. 23. HPF/FS. Dilated dictyosome cisternum (asterisk); intercisternal elements (arrowheads); flattened Golgi vesicles (arrow)

72 Dictyosomes of peripheral root cap cells secrete abundant mucilage, and in this process, cisternae become dilated and large vesicles are formed (Morr6 and Mollenhauer 1983). Compared to CF (Fig. 21), in high pressure frozen/freeze substituted peripheral cells: (1) Golgi vesicles were rounder, smoother, and much larger (Fig. 22), (2) coats were observed on Golgi vesicles of all sizes (Fig. 22), (3) intercisternal elements were better resolved both between Gotgi cisternae (Fig. 22) and Golgi associated vesicles (Fig. 23), and (4) contents of Golgi vesicles (Fig. 22) and dilated cisternae (Fig. 23) were more uniform in electron density. The trans Golgi network (Griffiths and Simons 1986), also known as the partially coated reticulum (Tanchak etal. 1988), was observed in both CF (not shown) and HPF/FS (Fig. 22).

J.Z. Kiss eta1.: Comparison of CF and HPF/FS of root tips osmotic activity, and does not penetrate across the plasmalemma (Dahl and Staehelin 1989). The dextran presumably suppresses extracellular ice crystallization yet does not disrupt the cells as penetrating cryoprotectant solutions such as glycerol or methanol can. Examination of Nicotiana and Arabidopsis root tips by light microscopy indicated that dextran at the concentration used did not appear to osmotically affect root tip cells in a deleterious manner after a 30 min exposure. In the present study, ice crystal damage was observed when distilled water was used as a freezing medium. While largely free of ice crystals, samples frozen in hexadecene (a non-penetrating, inert paraffin oil) were disrupted by numerous cracks and voids, effects presumably of high pressure induced shearing. Higher viscosity freezing media, such as dextrans, seem to be effective in controlling this problem.

Discussion

General comments on HPF/FS

Artifacts of HPF

Excellent ultrastructural preservation of entire Nicotiana and Arabidopsis root tips was obtained with HPF/ FS. These plant organs were cryofixed without ice crystal damage visible at the electron microscope level, and new details of cellular structure were observed. In tissues that were difficult to fix chemically, such as Nicotiana root tips, HPF/FS made adequate ultrastructural preservation possible. However, even in tissues that were relatively well-preserved by CF, such as Arabidopsis root tips (Sack and Kiss 1989), HPF/FS appeared to improve preservation e.g. new structures were observed such as coats around large Golgi vesicles.

Since there are relatively few published reports on HPF, it is important to identify artifacts that may be introduced by this technique. Craig and Staehelin (1988) described artifactual tears in the plasma membrane in material prepared by HPF/FF. We found, as did Kaeser et al. (1989), that in some cases HPF caused broken cell walls both at the periphery and within the root. Other possible artifacts oi~HPF, which were observed in some of the peripheral cells of Arabidopsis roots, were burst mucilage vesicles. Fortunately, such artifacts are easy to recognize, are confined to small areas, and the cytoplasm nearby can be well-preserved. Although HPF/FS results in excellent preservation of microtubules, nficrofilaments were not observed in the present study. Lancelle and Hepler (1989) have demonstrated that microfilament bundles can be well-preserved by HPF/FS. Although other techniques such as rhodamine-phalloidin have demonstrated that microfilaments are abundant in root cap cells (White and Sack 1990), HPF may not preserve individual microfilaments in these cells. It will be useful to try additional protocols, such as postfixation with uranyl acetate, to visualize microfilaments in high pressure frozen/freeze substituted root tips.

Effects of freezing media on quality of preservation In HPF, the specimen cup should be filled with a liquid medium to facilitate freezing (liquid improves thermal contact compared to air) and to prevent specimen damage that can result from pressure induced collapse of air bubbles in either the specimen or the cup (Craig and Staehelin 1988). We attribute the high yield of wellpreserved material to freezing the root tips in a dextran (MW38,800) solution. Draznin etal. (1988) also obtained better cryofixation with H P F of rat pituitary cells by freezing in dextran (MW 10,000) rather than in buffered saline solution. In the present study, approximately 80 % of the samples showed excellent preservation compared to 10-20% in a HPF/FF study in which samples were frozen in distilled water (Craig and Staehelin 1988). Dextran solutions are effective freezing media since dextran has a relatively large molecular weight and low

Comparison to other FS studies The significant advantage of HPF, compared to other ultrarapid freezing methods, for our study was that we were able to obtain good cryofixation of entire root tips (100-130 gm in diameter) without the use of permanent cryoprotectants. In many respects, the ultra-

J. Z. Kiss et al.: Comparison of CF and IIPF/FS of root tips structural parameters that characterize well-frozen biological specimens were comparable in this H P F study and in ultrarapid freezing studies o f smaller cells and tissues at atmospheric pressure. These parameters include (1) s m o o t h and turgid appearance o f m e m b r a n e systems (e.g., H o c h a n d H o w a r d 1980; HJppe 1985; Lancelle et al. 1986, 1987; Cresti et al. 1987); (2) closeappression o f the plasma m e m b r a n e to the cell wall (e.g., Cresti et al. 1987); (3) a dense cytoplasmic matrix (e.g., Lancelle etal. 1986); ( 4 ) i m p r o v e d preservation o f dictyosomes (e.g., H e a t h etal. 1985); (5) i m p r o v e d preservation o f microtubules (e.g., Lancelle etal. 1987, Tiwafi and Polito 1988) and crossbridges between microtubules (Lancelle etal. 1986); and (6) preservation o f vacuolar material (e.g., D a h m e n and H o b o r t 1986).

The E R and Golgi apparatus

I n H P F / F S , the E R lumen appeared to be filled with an electron dense material with a different texture f r o m that o f the b a c k g r o u n d cytoplasm. Perhaps this m a terial m a y represent newly synthesized proteins extracted by conventional techniques. H P F / F S also improved the ultrastructural preservation o f the Golgi apparatus in r o o t tip cells c o m p a r e d to CF. I n addition to relatively small, clathrin-coated vesicles visible in chemically-fixed material, H P F / F S revealed the presence o f larger Golgi vesicles with a c o a t lacking the m o r p h o l o g y typical o f ctathrin. A second class o f (non-clathrin) coated vesicles has been described in animal cells (Griffiths et al. 1985, Orci etal. 1986). M o r e study is needed to determine the composition and distribution o f coats associated with the plant Golgi apparatus. F u r t h e r implications o f the structural organization o f the Golgi apparatus as revealed by H P F / F S , such as discrete differences in staining patterns and width o f cis, medial, and trans cisternae, are discussed in a c o m p a n i o n paper (Staehelin et al. 1990). In conclusion, cryofixation has b e c o m e established as an i m p o r t a n t alternative to chemical fixation. This study demonstrates that H P F / F S is able to extend the advantages o f cryofixation to relatively large, intact plant tissues.

Acknowledgements

Financial support was provided by grants from Ohio State University (Office of Research and Graduate Studies), NASA ~AGW-780 to FDS), NIH (GM 18639 to LAS), and NSF (DCB 8615763 to LAS). We thank Dr. M. V. Parthasarathy (Cornell University) for helpful discussions.

73 References

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