TY - JOUR
AU - Zellnig,, Günther
AB - Abstract Different fixation protocols [chemical fixation, plunge and high pressure freezing (HPF)] were used to study the effects of Zucchini yellow mosaic virus (ZYMV) disease on the ultrastructure of adult leaves of Styrian oil pumpkin plants (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) with the transmission electron microscope. Additionally, different media were tested for freeze substitution (FS) to evaluate differences in the ultrastructural preservation of cryofixed plant leaf cells. FS was either performed in (i) 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate, (ii) 0.01% safranin in anhydrous acetone, (iii) 0.5% glutaraldehyde in anhydrous acetone or (iv) anhydrous acetone. No ultrastructural differences were found in well-preserved cells of plunge and high pressure frozen samples. Cryofixed cells showed a finer granulated cytosol and smoother membranes, than what was found in chemically fixed samples. HPF led in comparison to plunge frozen plant material to an excellent preservation of vascular bundle cells. The use of FS-media such as anhydrous acetone, 0.01% safranin and 0.5% glutaraldehyde led to low membrane contrast and did not preserve the inner fine structures of mitochondria. Additionally, the use of 0.5% glutaraldehyde caused the cytosol to be fuzzy and partly loosened. ZYMV-induced ultrastructural alterations like cylindrical inclusions and dilated ER-cisternae did not differ between chemically fixed and cryofixed cells and were found within the cytosol of infected leaf cells and within sieve tube elements. The results demonstrate specific structural differences depending on the FS-medium used, which has to be considered for investigations of selected cell structures. freeze substitution, Cucurbita pepo, high pressure freezing, transmission electron microscopy, Zucchini yellow mosaic virus Introduction Analyses with the transmission electron microscope (TEM) are directly linked with the fixation of the investigated tissue. The most commonly used fixation method for TEM is chemical fixation. The reason therefore is the adequate preservation of many cellular components including enzymes, the clarity of structural details and the ease and lower costs of its application. However, the relatively long fixation times of chemical fixation in combination with dehydration can lead to several artefacts including distorted membranes and organelles [1–6]. Cryofixation is one powerful alternative to chemical fixation. However, most methods of cryofixation require expensive equipment and are technically challenging. At the moment there are a number of different methods available to cryofix biological samples like plunge freezing, cold metal block freezing or high pressure freezing (HPF). Since biological samples are frozen ultrarapidly in milliseconds by using these methods ultrastructural alterations, which may happen during chemical fixation, like swelling, shrinkage etc. are avoided [7] and the cell morphology is kept closer to its living state because soluble cell constituents are maintained [4,8]. One disadvantage of cryofixation is that most procedures cannot efficiently freeze the large amount of water within the samples, especially in the deeper layers. Ice crystal damage is therefore the main reason for dissatisfying freezing results of plant samples [7] especially of adult leaves, containing large vacuoles filled with mostly water. In comparison to unicellular organisms, protoplasts and animal tissues there are only few protocols available for the successful cryofixation of plant tissues like leaves [9–13]. The severity of ice crystal damage strongly depends on the cryofixation technique used. The most effective approach of cryofixation is HPF, which allows the preservation of plant samples up to 200 μm from its surface [14,15]. Due to the high costs of the equipment, the usage of this method is still very limited, especially in plant sciences [11]. The most widely used cryofixation procedure is plunge freezing, which is the simplest and least expensive method. It only allows freezing of the plant sample to a depth of 1–50 μm and, therefore, the amount of well-preserved cells is far lower in comparison to HPF [16]. After cryofixation, samples need to go through freeze substitution (FS) and resin embedding where the solidified water is (i) replaced by an anhydrous solvent at temperatures precluding ice crystal growth and subsequently by (ii) plastic resins, which allow the sectioning of the embedded material. Various different protocols are available for FS and their results vary greatly depending on the substitution media and plant material used. The most commonly used FS-medium is osmium tetroxide in anhydrous acetone containing low concentrations of uranyl acetate. This FS-medium usually leads to an appropriate ultrastructural preservation of the cytosol and organelles [4,10,16–20]. To avoid negative effects of osmium tetroxide such as a loss of the antigenicity of cell constituents [21] a number of different fixatives are used during FS. The application of pure acetone alone with or without low concentrations of aldehydes with or without uranyl acetate has been proven to preserve the ultrastructure and antigenicity of cells [5,18,19], as well as the use of safranin [22], which is commonly used as a staining agent in light microscopy. However, by using the above described FS-media, differences were noticed in the preservation of cell structures. In the present study we investigated the impacts of different FS-media on the ultrastructure of adult leaves of virus-infected and control Styrian oil pumpkin plants (Cucurbita pepo L. subsp. pepo var. styriaca Greb.). Since 1997 severe epidemics of Zucchini yellow mosaic virus (ZYMV)-disease have been causing crop loss in Styrian oil pumpkin production of up to 60% every year. ZYMV belongs to the potyvirus group and causes severe ultrastructural alterations within the plant including cylindrical inclusions (CI) and proliferated endoplasmatic reticulum (ER) [23]. In a previous study we investigated the impact of ZYMV-disease on the ultrastructure of chemically fixed leaves from Styrian oil pumpkin plants [24]. One typical ultrastructural alteration found during ZYMV-infection was the occurrence of dilated ER-cisternae. However, it was not clear, whether those changes were induced by the virus or a result of chemical fixation of the tissue since it has been shown that chemical fixation can lead to dramatic differences in cellular ultrastructure, especially of membranous structures, in comparison to cryofixation methods [6]. Therefore, to obtain unambiguous data of ultrastructural changes during ZYMV-infection, well-established chemical- (glutaraldehyde/osmium fixation) and cryofixation techniques (HPF, plunge freezing) were used in the present study in order to compare and complete the previously obtained data of ultrastructural changes during ZYMV-infection in leaves of Styrian oil pumpkin plants. To our knowledge this is the first time that potyvirus-induced ultrastructural modifications within leaves have been studied using different methods of cryofixation (plunge freezing and HPF) and were then compared with chemically fixed cells. Different FS-media were additionally used to obtain more information about the ultrastructural preservation of cell structures in adult plant leaves after cryofixation. Materials and methods Plant material Seeds of Styrian oil pumpkin received from Saatzucht Gleisdorf (Plant Breeding Company, Gleisdorf, Austria) were germinated on a humid Perlite cloth. After 1 week seedlings were replanted in pots and transferred into separated growth chambers with a photoperiod of 12 h (PAR 400–700 μmol m−2 s−1). Day and night temperatures were 22 and 18°C, respectively; the relative humidity was 70%. The plants were kept at 100% relative water content. Cotyledons of one group were infected with the sap of ZYMV-infected plant material before the first foliage leaves emerged. One gram of infected plant material (leaves or mesocarp) of Styrian oil pumpkin plants was homogenated in 1 ml of a 1%-K2HPO4 solution (pH 6.8). After applying celite (Sigma) to the homogenate, the inoculum was spread out on the cotyledons of the seedlings of one group. Mock inoculation was performed on control plants. Three weeks post inoculation different samples of fully developed adult leaves (fifth leaf counted from the bottom of the plants) of both control and infected plants were harvested separately. Chemical fixation Small pieces (∼1.5 mm2) of control and ZYMV-infected leaves were cut in a drop of 3%-glutaraldehyde in 0.06 M Sørensen phosphate buffer (pH 7.2) on a modelling wax plate, transferred into glass vials and fixed in the solution for 90 min at room temperature (RT). After rinsing the material 5 times for 10 min each in buffer at RT, postfixation was accomplished in 1%-osmium tetroxide solved in 0.06 M Sørensen phosphate buffer for 90 min at RT. The samples were then rinsed in buffer (4 times for 15 min each) and dehydrated in a graded acetone series (50, 70, 90 and 100%) for 2 times for 10 min at each step. Pure acetone was then replaced with propylene oxide and the specimen was gradually infiltrated with increasing concentrations of Agar 100 epoxy resin (30, 50, 70 and 100%) mixed with propylene oxide for a minimum of 3 h for each step. Samples were finally embedded in pure, fresh Agar 100 epoxy resin and polymerized at 60°C for 48 h. Ultrathin sections (80 nm) were cut with a Reichert Ultracut S microtome, post-stained with lead citrate (5 min) followed by uranyl acetate (15 min) and observed in a Philips CM10 TEM. HPF and FS Small pieces (1.2 mm in diameter) of ZYMV-infected and control leaves were punched out with a punching device for flat specimen carriers from Leica Microsystems (Vienna, Austria) in a drop of 1-hexadecane on a soft piece of rubber. Subsequently the specimens were transferred into the cavity (1.2 mm in diameter) of gold plated flat specimen carriers which were 200 μm in depth and pre-filled with 1-hexadecene as a cryoprotectant. With the help of a loading station the specimen carriers were tightened securely into the pod of flat specimen holders, which were tightened to the loading device, inserted and subsequently high pressure frozen in the Leica EM Pact (Leica Microsystems; for technical information see Studer et al. [11]). Platelets containing frozen samples were then transferred and stored under liquid nitrogen conditions in transfer boxes or pre-chilled specimen baskets. For FS specimens were transferred into pre-cooled cryogenic vials (Corning Inc., Corning, NY) filled with FS-media consisting of either (i) 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate, (ii) 0.01% safranin O in anhydrous acetone (iii) 0.5% glutaraldehyde in anhydrous acetone or (iv) anhydrous acetone. FS was carried out in a deep freezer at −80°C (72 h), −65°C (24 h), −30°C (24 h), 0°C (12 h) and RT (1 h). After the samples were rinsed twice in anhydrous acetone for 15 min, they were infiltrated by solutions that contained acetone and Agar 100 epoxy resin in mixtures (2:1, 1:1, 1:2) and pure epoxy resin for at least 3 h, each step at RT. The embedded samples were then polymerized in pure, fresh Agar 100 epoxy resin for 48 h at 60°C. Ultrathin sections were cut, post-stained, as described above, and observed in the TEM. Plunge freezing Plunge freezing was performed with a Leica KF 80 immersion cryofixation system (Leica Microsystems). Parts of control and ZYMV-infected leaves (∼1.5 mm in diameter) were cut on a modelling wax plate in unbuffered 0.3 M sucrose which acted as a cryoprotectant. Subsequently, samples were attached to the broad end of a thin, T-shaped stripe of filter paper. After sucking off the excess of fluid from the samples, the longer end of the filter paper was clamped into the tweezers of the Leica KF 80 immersion cryofixation system while the other, broader end carrying the specimen, was plunged into liquid propane, which was cooled with liquid nitrogen to constant freezing temperature at −170°C. Frozen samples still attached to the stripes of the T-shaped filter paper were then transferred under liquid nitrogen conditions into pre-chilled specimen baskets where they were stored. For FS filter papers still holding the specimen were transferred under liquid nitrogen into pre-cooled cryogenic vials filled with the substitution reagent containing 2% osmium tetroxide and 0.2% uranyl acetate in anhydrous acetone. FS and embedding were carried out as described above for the high pressure frozen plant material. Results To obtain unambiguous data about ZYMV-induced ultrastructural changes within Styrian oil pumpkin plants, special freezing techniques (plunge freezing, HPF) and different methods of FS were used to compare the ultrastructure of both chemically and cryofixed ZYMV-infected and control leaf cells. Generally, no differences were found in the preservation of ZYMV-induced ultrastructural alterations between chemically and cryofixed cells. The virus induced CI and pinwheels as well as dilated ER-cisternae often forming vesicles containing granulated material at its end. However, differences were found in the structural appearance of cellular components depending on the type of fixation and FS-media used. The amounts of well-preserved cells in chemically fixed leaves (Fig. 1a) were by far higher than in cryofixed cells, allowing time saving preparations and investigations of the specimen. Fig. 1 Open in new tabDownload slide Micrographs of mesophyll cells from leaves of Styrian oil pumpkin plants fixed with different methods. Bars: 1 µm (Insets: 200 nm). (a) Chemically fixed ZYMV-infected cells with a dense cytosol showing CI (asterisk), dilated-ER cisternae (arrows), parts of a nucleus (N), mitochondrion (M), chloroplast (C) with starch (St) and plastoglobulus. The plasmalemma appears undulated (arrowheads). Inset shows dilated ER forming vesicles containing granulated material at its end (arrow) (b) Plunge frozen control cells after FS in 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate with chloroplasts (C) containing starch (St) and plastoglobuli, mitochondria (M), and dictyosomes (D). Inset shows ER from a control cell after plunge freezing. High pressure frozen cells that were freeze substituted in 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate from (c) leaves of control plants containing a nucleus (N) with a dilated envelope (asterisk), chloroplast (C) with electron translucent plastoglobuli, mitochondria (M), smooth plasmalemma (arrowheads), ER (arrow) and plasmodesmata (Pd) within the cell walls and (d) from ZYMV-infected leaf-material showing a chloroplast (C) with thylakoids and some plastoglobuli, mitochondria (M), a dictyosome (D), cylindrical inclusions (arrowhead) and dilated ER (arrow). Inset shows dilated ER forming vesicles containing granulated material at its end (arrow). V, vacuoles; IS, intercellular spaces. Fig. 1 Open in new tabDownload slide Micrographs of mesophyll cells from leaves of Styrian oil pumpkin plants fixed with different methods. Bars: 1 µm (Insets: 200 nm). (a) Chemically fixed ZYMV-infected cells with a dense cytosol showing CI (asterisk), dilated-ER cisternae (arrows), parts of a nucleus (N), mitochondrion (M), chloroplast (C) with starch (St) and plastoglobulus. The plasmalemma appears undulated (arrowheads). Inset shows dilated ER forming vesicles containing granulated material at its end (arrow) (b) Plunge frozen control cells after FS in 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate with chloroplasts (C) containing starch (St) and plastoglobuli, mitochondria (M), and dictyosomes (D). Inset shows ER from a control cell after plunge freezing. High pressure frozen cells that were freeze substituted in 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate from (c) leaves of control plants containing a nucleus (N) with a dilated envelope (asterisk), chloroplast (C) with electron translucent plastoglobuli, mitochondria (M), smooth plasmalemma (arrowheads), ER (arrow) and plasmodesmata (Pd) within the cell walls and (d) from ZYMV-infected leaf-material showing a chloroplast (C) with thylakoids and some plastoglobuli, mitochondria (M), a dictyosome (D), cylindrical inclusions (arrowhead) and dilated ER (arrow). Inset shows dilated ER forming vesicles containing granulated material at its end (arrow). V, vacuoles; IS, intercellular spaces. For the most part the ultrastructure of well-preserved cryofixed cells did not differ between high pressure and plunge frozen plant cells (Fig. 1b–d). Nevertheless, within plunge frozen leaves only single cells of the epidermis and the upper quarter of the first mesophyll cell layer, which were plunged into liquid propane first, were well-preserved (up to a depth of 30–40 μm) and could be used for ultrastructural investigations. Ice crystal damages and ultrastructural alterations due to insufficient fixation were observed commonly in epidermal cells, and got more evident in the mesophyll cell layer. Disruptions or cracks within membranes were observed frequently; however, only cells without such alterations were investigated (Fig. 1b). In high pressure frozen leaf-material, cells of the epidermis and the first mesophyll layer (depth of ∼50–70 μm) were well-preserved without ice crystal damage (Figs 1 and 3). However, disruptions or cracks within cell walls and membranes were observed. Cells of vascular bundles which occurred at a depth of ∼100 μm were generally well-preserved although the surrounding mesophyll cells showed disruptions or cracks within membranes and cell walls or other ultrastructural damage due to poor cryofixation (Fig. 2). Fig. 2 Open in new tabDownload slide Micrographs of high pressure frozen vascular bundle cells from leaves of Styrian oil pumpkin plants. Bars: (a), 1 µm; (b), 4 µm. FS was carried out in 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate. (a) Sieve tube element from ZYMV-infected leaves containing cylindrical inclusions (arrows) and companion cells with mitochondria (M), plastid (P), and cylindrical inclusions (arrow) within the dense cytosol. (b) Overview of parts of a vascular bundle from control leaves with companion cells containing mitochondria (M), plastids (P), a nucleus (N), sieve tube elements (STE) and a xylem element (XE). Ultrastructural damages like disruptions or cracks within cell walls and membranes were found in surrounding mesophyll cells (arrows). V, vacuoles. Fig. 2 Open in new tabDownload slide Micrographs of high pressure frozen vascular bundle cells from leaves of Styrian oil pumpkin plants. Bars: (a), 1 µm; (b), 4 µm. FS was carried out in 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate. (a) Sieve tube element from ZYMV-infected leaves containing cylindrical inclusions (arrows) and companion cells with mitochondria (M), plastid (P), and cylindrical inclusions (arrow) within the dense cytosol. (b) Overview of parts of a vascular bundle from control leaves with companion cells containing mitochondria (M), plastids (P), a nucleus (N), sieve tube elements (STE) and a xylem element (XE). Ultrastructural damages like disruptions or cracks within cell walls and membranes were found in surrounding mesophyll cells (arrows). V, vacuoles. Well-preserved cells of both cryofixation techniques showed smoother cellular membranes of plasmalemma, tonoplast and organelles without undulation in comparison to chemically fixed cells (Fig. 1). Membranes of chemically fixed cells were well preserved and heavily stained (Fig. 1a). Cryofixation and FS with 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate resulted in well preserved and stained membranes similar to those found in chemically fixed cells (Fig. 1b–d). FS in 0.01% safranin in anhydrous acetone, 0.5% glutaraldehyde in anhydrous acetone, and anhydrous acetone often resulted in rather poorly stained membranes, which were often difficult to distinguish from the surrounding cell structures (Figs 3b–d and 4). Fig. 3 Open in new tabDownload slide Micrographs of mesophyll cells from high pressure frozen leaves of Styrian oil pumpkin plants freeze substituted in different media. Bars: (a) = 2 µm; (b–d) = 1 µm. (a) Overview of mesophyll cells from ZYMV-infected leaves freeze substituted in 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate showing chloroplasts (C), mitochondria (M), peroxisomes (Px), nuclei (N) and vacuoles (V). (b) Control cell freeze substituted in 0.5% glutaraldehyde in anhydrous acetone containing chloroplasts (C) with thylakoids and plastoglobuli, and a mitochondrion (M). (c) ZYMV-infected cell after FS in 0.01% safranin in anhydrous acetone showing chloroplasts containing starch (St) and heavily dilated thylakoids. Within the dense cytosol cylindrical inclusions (arrowhead) and dilated-ER cisternae (arrows) occurred. (d) ZYMV-infected cells freeze substituted in anhydrous acetone with chloroplasts (C) containing starch (St) and a well-defined thylakoid system, a nucleus (N), cylindrical inclusions (arrowhead) and dilated ER-cisternae (arrows) within the dense cytosol. V, vacuoles; IS, intercellular spaces. Fig. 3 Open in new tabDownload slide Micrographs of mesophyll cells from high pressure frozen leaves of Styrian oil pumpkin plants freeze substituted in different media. Bars: (a) = 2 µm; (b–d) = 1 µm. (a) Overview of mesophyll cells from ZYMV-infected leaves freeze substituted in 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate showing chloroplasts (C), mitochondria (M), peroxisomes (Px), nuclei (N) and vacuoles (V). (b) Control cell freeze substituted in 0.5% glutaraldehyde in anhydrous acetone containing chloroplasts (C) with thylakoids and plastoglobuli, and a mitochondrion (M). (c) ZYMV-infected cell after FS in 0.01% safranin in anhydrous acetone showing chloroplasts containing starch (St) and heavily dilated thylakoids. Within the dense cytosol cylindrical inclusions (arrowhead) and dilated-ER cisternae (arrows) occurred. (d) ZYMV-infected cells freeze substituted in anhydrous acetone with chloroplasts (C) containing starch (St) and a well-defined thylakoid system, a nucleus (N), cylindrical inclusions (arrowhead) and dilated ER-cisternae (arrows) within the dense cytosol. V, vacuoles; IS, intercellular spaces. Fig. 4 Open in new tabDownload slide Micrographs of mesophyll cells from high pressure frozen leaves of Styrian oil pumpkin plants. Bars: 1 µm. (a) Close up of a control cell after FS in anhydrous acetone with a chloroplast (C) containing a well-defined thylakoid system, and a plastoglobulus. (b) Close up of a ZYMV-infected cell freeze substituted in 0.01% safranin in anhydrous acetone showing a chloroplast (C) with heavily dilated thylakoids and some plastoglobuli. Fig. 4 Open in new tabDownload slide Micrographs of mesophyll cells from high pressure frozen leaves of Styrian oil pumpkin plants. Bars: 1 µm. (a) Close up of a control cell after FS in anhydrous acetone with a chloroplast (C) containing a well-defined thylakoid system, and a plastoglobulus. (b) Close up of a ZYMV-infected cell freeze substituted in 0.01% safranin in anhydrous acetone showing a chloroplast (C) with heavily dilated thylakoids and some plastoglobuli. Generally, the cytosol of both cryofixed and chemically fixed cells was dense containing well-preserved dictyosomes, ER-cisternae, vesicles and other organelles (Fig. 1). Nevertheless, the cytosol of chemically fixed cells appeared roughly textured in comparison to cryofixed cells, which had a denser and more finely granulated cytosol (Fig. 1). FS with 0.5% glutaraldehyde in anhydrous acetone did not preserve the cytosol as well as the other FS-media. The cytosol was fuzzy and partly loosened whereas the organelles remained unaffected (Fig. 3b). CI were found, which occurred as bundles if cut longitudinally and as scrolls and pinwheels if cut transversely, within the cytosol of ZYMV-infected cells of the epidermis, mesophyll, phloem parenchyma, companion cells and sieve tube elements (Figs 1a, 1d, 2a, 3c and 3d). No differences were found in the ultrastructural appearance between cryofixed and chemically fixed cells. Additionally, ZYMV-infection induced dilated ER, especially at its end (Fig. 3d), often forming vesicles containing granulated material, throughout the cytosol of infected leaf cells, which was found in both cryofixed and chemically fixed ZYMV-infected cells (Figs 1a and 1d) but not in the controls (Figs 1b and 1c). ZYMV-particles were observed throughout the cytosol of cryofixed and chemically fixed cells and did not show structural differences. Chemically fixed and cryofixed chloroplasts appeared spherical, longish or amoeboid without protuberances (Figs 1, 3 and 4). The envelope of chemically fixed chloroplasts was heavily stained and slightly undulated. The chloroplast envelope of cryofixed cells was smooth but generally only clearly visible when cells were freeze substituted in media containing 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate (Figs 1b and 1d). Chloroplasts contained dense stroma, plastoglobuli and well-preserved thylakoids organized as grana stacks or single membranous structures. It was evident that cells that were freeze substituted with 0.5% glutaraldehyde in anhydrous acetone showed few dilated thylakoids (Fig. 3b), whereas FS with 0.01% safranin led to heavily dilated thylakoids (Figs 3c and 4b). The thylakoid-membranes were well stained and clearly distinguishable from the stroma, except when cells were freeze substituted in 0.01% safranin in anhydrous acetone, 0.5% glutaraldehyde in anhydrous acetone, and anhydrous acetone (Figs 3b, 3d and 4). In chloroplasts of those cells, membranes of thylakoids often appeared faded and were only distinguishable if they were separated by darker stained regions of stroma (Figs 3d and 4a), or were dilated (Figs 3b, 3c and 4b). Generally, thylakoids appeared electron translucent in all chloroplasts of cryofixed cells in contrast to chemically fixed cells where thylakoids were stained dark. Plastoglobuli appeared electron opaque in chemically fixed cells and electron translucent in chloroplasts of cryofixed cells. Starch grains were frequently observed within chloroplasts. In cryofixed cells they showed no signs of shrinkage and were closely associated with the stroma (Figs 1b, 3c and d), whereas in chemically fixed chloroplasts they were shrunken resulting in electron translucent, stroma-free regions around the actual starch grains (Fig. 1a). Mitochondria of cryofixed cells had a smooth, not undulated envelope, whereas the membranes of mitochondria in chemically fixed cells often appeared to be wrinkled and undulated (Fig. 1). The membranes (including cristae) of mitochondria were only clearly visible when cells were chemically fixed or freeze substituted in 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate (Fig. 1). When cells were fixed with the other FS-media, membranes of mitochondrial were only partially visible and the mitochondrial matrix appeared to be dense (Fig. 3b). Therefore, with such FS-media, a clear differentiation between mitochondria and peroxisomes was not always possible, and if possible, it was only based on characteristic forms of mitochondria and/or size (Fig. 3b). In cryofixed samples the envelopes of nuclei appeared smooth and the membranes were further apart from each other (Figs 1c and 3a) than what was observed in chemically fixed cells (Fig. 1a). The perinuclear space in cryofixed samples had an average width of ∼35 nm on the section in comparison to 20 nm in chemically fixed specimen. The nucleoplasm of chemically fixed cells was more roughly granulated (Fig. 1a) than in nuclei of high pressure frozen cells (Figs 1c, 3a and 3d). Hetero- and euchromatic regions were clearly separated in both chemically and cryofixed cells. Dictyosomes exhibited more detailed structures in cryofixed cells compared to chemically fixed cells. Each cisterna was more distinctively separated from the other and vesicles were clearly visible (Figs 1b and 1d). Discussion In the present study we investigated differences in the ultrastructural preservation of chemically and cryofixed ZYMV-infected and control leaves in their adult stage. We tested the advantages and disadvantages of four different FS-media on plunge and high pressure frozen leaf-material and compared them with chemically fixed leaf cells. No ultrastructural differences were found between well-preserved cells of plunge frozen and high pressure frozen leaves. Therefore, both methods are suitable for studying the ultrastructure of plant material as close to its living state as possible [16]. Nevertheless, due to ice crystal damage and insufficient freezing rates of plunge frozen leaves, only cells of the epidermis and the upper quarter of the first mesophyll cell layer could be used for ultrastructural investigations. Additionally, a large number of samples needed to be sectioned in order to obtain a representative number of well-preserved cells. HPF resulted in a considerably larger number of well-preserved cells within one sample. Well-preserved high pressure frozen cells were found in the epidermis, first mesophyll cell layers and also within vascular bundle cells. Considering these differences HPF is much better suited for ultrastructural studies of the present plant leaf-material under the described conditions. HPF has also been demonstrated to be the method of choice for other plant samples especially for thick tissues [8,11] like the investigated adult leaves which contain distinct vacuoles. However, considering possible negative effects of HPF like the influence of high pressure on the ultrastructure, the most suitable cryofixation method should be separately evaluated for each plant material by using both HPF and plunge freezing [3,16,25]. In the present study the preparation depth of 200 μm as reported in the literature for good ultrastructural preservation after HPF [14,15] was not achieved for adult Cucurbita leaves. High water content of leaf cells due to large vacuoles or the high number of different trichomes on the surface of the leaves might have been the reason for the dissatisfying freezing results below a depth of ∼70 μm after HPF. The excellent preservation of xylem and phloem cells makes HPF a powerful tool to study the ultrastructure of vascular bundle cells as close to their native state as possible [16,26–29]. It seems that the cryogen enters and flows through the vascular bundles (most probably through xylem cells and sieve tube elements) due to the high pressure of injection and cryofixes the surrounding cells. This mechanism is supported by the fact that vascular bundle cells of plunge frozen leaf-material were not well-preserved and were unsuitable for ultrastructural investigations. Within chemically fixed leaves, the amount of well-preserved cells remained larger than in those of cryofixed leaves. Various differences were found between cells of chemically and cryofixed samples. Chemically fixed cells showed undulated membranes of the tonoplast, the plasmalemma and the envelopes of organelles, whereas membranes of cryofixed cells were smooth. Especially the plasmalemma, which was closely aligned to the cell walls in cryofixed cells, appeared slightly detached and wrinkled in chemically fixed cells. Such differences in the preservation quality of membranes are typical between chemically and cryofixed samples due to the fact that chemical fixatives disrupt the membranes by their slow penetration, and induce osmotic stress and fragmentation of delicate membrane networks [4,6,29,30]. Membranes within organelles were, in comparison to the envelope of organelles, well-preserved and smooth in chemically fixed and cryofixed samples. Besides differences in electron opacity (electron opaque stained in chemically fixed chloroplasts and electron translucent in cryofixed chloroplasts), no differences in the ultrastructure of thylakoids were observed in chloroplasts of well-preserved chemically and cryofixed cells. However, cells, which were high pressure frozen and freeze substituted with 0.01% safranin in anhydrous acetone, showed heavily dilated thylakoids. FS-media, such as anhydrous acetone, 0.01% safranin and 0.5% glutaraldehyde, did not preserve the fine structures of mitochondria. Those FS-media should therefore not be used for detailed investigations of the ultrastructure of chloroplasts and mitochondria, respectively. Nevertheless, safranin has been successfully used as FS-medium to study the ultrastructure of cryofixed barley and pea leaves and has been proven to preserve the antigenicity of the sample if specimens are embedded in Lowicryl at low temperatures [22]. The authors reported a very good ultrastructural preservation rendering the use of further fixatives like osmium tetroxide and glutaraldehyde unnecessary. FS-media such as pure acetone alone or supplemented with uranyl acetate with and without formaldehyde have also been shown to yield satisfactory ultrastructural preservation as well as labelling specificity and density in Ledebouria socialis pollen grains and anthers tissues embedded in LR-White [19]. However, information about the preservation of these FS-media after HPF in leaf-tissues is rare [31]. Generally, membranes of cryofixed cells that were freeze substituted with 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate were well stained, and comparable to the staining of membranes in chemically fixed cells. It has been demonstrated previously that membranes involved in viral replication were completely unstained after FS in 2% osmium and 0.1% uranyl acetate in acetone [32]. Yet, in the present study no difference was observed in the staining intensity of control or ZYMV-infected cell membranes, which also were freeze substituted in 2% osmium and 0.2% uranyl acetate in anhydrous acetone. Therefore such effects of ZYMV-infection on membranes can be excluded from the present plant microbe interaction. Low membrane contrast in the present study was only found when cells were freeze substituted in FS-media such as anhydrous acetone, 0.01% safranin and 0.5% glutaraldehyde but not when 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate was used as FS-medium. Insufficient contrast of biological membranes in cryofixed samples is most probably due to the extraction of lipids during FS, failure to bind an electron-dense fixative such as osmium, or failure to bind post-embedding stains [18]. These effects can be avoided, for example, by adding water to the FS-media [33], raising the temperature of osmium tetroxide in acetone to 40°C [34], combining glutaraldehyde and osmium in the initial FS step [35] and others [18]. Nevertheless, in the present study FS in 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate led to a very good preservation and staining of membranes rendering the above described methods unnecessary in the present experimental design. The cytosol was preserved well in both chemically fixed and cryofixed cells. Cryofixed cells showed an even finer granulated cytosol than chemically fixed cells where cytoplasmic proteins often aggregate and are extracted from the cytosol [5,36]. FS with 0.5% glutaraldehyde in anhydrous acetone caused the cytosol to be partly loosened, yet without negative effects on organelles. These results demonstrate that low concentrations of glutaraldehyde alone are not well suited as FS-media if the cytosol is the main centre of interest of ultrastructural investigations. Nevertheless, it has been shown that glutaraldehyde, especially in combination with low concentrations of uranyl acetate, leads to an adequate preservation of the ultrastructure and antigenicity of various specimens [5,18]. ZYMV-induced CI and dilated ER-cisternae were found within the cytosol of both chemically and cryofixed leaf cells. These structures did not differ between chemically and cryofixed cells and were similar to what was already described in a previous work [24]. Therefore dilated ER-cisternae can be regarded as real ZYMV-induced stress responses of the cell and are not a result of chemical fixation. CI were also found in cryofixed sieve tube elements. CI are formed by the virus CI-protein, which accumulates within the cytosol of infected cells [37–39] and are involved in virus replication, protein synthesis and short distance movement of the virus from cell-to-cell [40–43]. Nevertheless, the functions and mechanisms behind the accumulation of CI in sieve tube elements are not clear since they do not support virus replication or protein synthesis due to the absence of nuclei. Therefore, it seems that CI-proteins are imported into sieve tube elements from neighbouring cells (e.g. companion cells) where they are assembled into CI. Within sieve tube elements, CI could be involved in the movement of the virus. Chloroplasts were preserved well in both chemically fixed and cryofixed cells. Protuberances of plastids (stromules) as observed (i) by GFP-fluorescence in Nicotiana [44] or Lycopersicon [45], (ii) after HPF in rice leaves [10] and (iii) after chemical fixation in Ranunculus [46], Acetabularia [47] and Spinacia [48] were not found in control and ZYMV-infected leaves in the present investigations. It has been proposed previously that stromule formation might also be induced by various biotic and abiotic stress situations [44]. Nevertheless, during the present experiments, ZYMV-infection as a biotic stress factor, did not induce the formation of stromules. Well-preserved nuclei have been found in various high pressure frozen plant material [4,5,11,27,28]. In the present study, no obvious differences in the ultrastructural preservation of nuclei were found between chemically and cryofixed samples. A differentiation between hetero- and euchromatin was possible in chemically fixed, high pressure and plunge frozen cells. However, in cryofixed samples the nucleoplasm appeared more finely granulated and the perinuclear space was dilated when compared to chemically fixed specimen. Nevertheless, it is not clear at the moment whether these differences are due to fixation artefacts or represent the natural state. Concluding remarks Well-preserved plunge and high pressure frozen plant leaf-material showed similar ultrastructural cell characteristics and are therefore both well suited for cryofixation. Due to the higher amounts of well-preserved cells (up to 70 μm in depth) in high pressure frozen samples in comparison to plunge frozen ones, HPF can be recommended for the study of the ultrastructure of adult plant tissues with distinct vacuoles, like the investigated leaves. Since HPF also led to a very good preservation of vascular bundle cells, it can be used if phloem or xylem cells are the main object of interest. Various ultrastructural differences were observed between well-preserved chemically fixed and cryofixed samples including the conservation of membranes, the cytosol and organelles. However, these differences are only important if membranes are the main objects of interest. FS-media containing osmium and uranyl acetate led to a similar preservation of cryofixed cells (membranes, organelles etc.) than what was observed in chemically fixed cells. This FS-medium can therefore be recommended if the ultrastructure of cryofixed and chemically fixed cells are compared or connected with investigations on dynamic, fast running cellular processes. FS-media containing safranin, glutaraldehyde or anhydrous acetone can be used to obtain suitable ultrastructural preservation if FS-media, without osmium, have to be used. This work was supported by the Austrian Science Fund, Project no. P16273-B06. 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TI - Effects of different fixation and freeze substitution methods on the ultrastructural preservation of ZYMV-infected Cucurbita pepo (L.) leaves
JF - Journal of Electron Microscopy
DO - 10.1093/jmicro/dfi054
DA - 2005-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/effects-of-different-fixation-and-freeze-substitution-methods-on-the-I9ZDR8qOKh
SP - 393
EP - 402
VL - 54
IS - 4
DP - DeepDyve
ER -