Palaeontological evidence for membrane fusion between a unit membrane and a half-unit membrane

Abstract

Membrane fusion is of fundamental importance for many biological processes and has been a topic of intensive research in past decades with several models being proposed for it. Fossils had previously not been considered relevant to studies on membrane fusion. But here two different membrane fusion patterns are reported in the same well-preserved fossil plant from the Miocene (15–20 million years old) at Clarkia, Idaho, US. Scanning electron microscope, transmission electron microscope, and traditional studies reveal the vesicles in various states (even transient semi-fusion) of membrane fusion, and thus shed new light on their membrane structure and fusion during exocytoses. The new evidence suggests that vesicles in plant cells may have not only a unit membrane but also a half-unit membrane, and that a previously overlooked membrane fusion pattern exists in plant cells. This unexpected result from an unexpected material not only marks the first evidence of on-going physiological activities in fossil plants, but also raises questions on membrane fusion in recent plants.

• Membrane fusion
• half-unit membrane
• fossil
• plant

Introduction

Membrane fusion has been a focus of intensive biological and biophysical research in past decades (Palade & Bruns 1968, Poole et al. 1970, Lucy 1970, Satir et al. 1973, Chandler & Heuser 1979, Scherz et al. 1990, Galway et al. 1993, Robinson et al. 1996, Thiel et al. 1998, Thiel & Battey 1998, Battey et al. 1999, Blatt et al. 1999, Homann & Thiel 1999, Blatt 2002, Gruner 2002, Yang & Huang 2002, Hed & Safran 2003, Malinin & Lentz 2004, Ostrowski et al. 2004, Giraudo et al. 2005, Lu et al. 2005, Chernomordik & Kozlov 2005, Ungermann & Langosch 2005, Anderson 2006, Koumandou et al. 2007, Wong et al. 2007) because it is closely related to many fundamental biological processes and medical applications, including cell signaling, cell growth, development, homeostasis, stomatal function, secretion, cell wall formation, fertilization, product transport, drug delivery, and gene therapy (van der Woude et al. 1971, Chandler & Heuser 1979, Meindl et al. 1992, Galway et al. 1993, Thiel et al. 1998, Battey et al. 1999, Homann & Thiel 1999, Blatt 2002, Yang & Huang 2002, Chernomordik & Kozlov 2005, Wong et al. 2007). Biological membranes, including vesicular ones, are all thought to have a double-layered structure (Kimball 1970, Ambrose & Easty 1979, Zheng 1996, Staehelin & Newcomb 2002, Chernomordik & Kozlov 2005) even though half-unit membranes have been reported several times in cells (Hodge et al. 1956, Csillik 1963, Cardell et al. 1967, Anderson et al. 1970, Yatsu et al. 1971, Konar et al. 1972, Yatsu & Jacks 1972, Gardner & Hess 1977, Hilhorst et al. 1982, de Castro & Martinez-Honduvilla 1984, Scherz et al. 1990, Somerville et al. 2002, Schumann et al. 2003). Although vesicle trafficking is of essential importance for nearly all eukaryotic cells (Blatt et al. 1999, Blatt 2002, Koumandou et al. 2007) and several models and modifications have been proposed for the fusion between vesicle and cytoplasmic membranes (Thiel & Battey 1998, Blatt et al. 1999, Battey et al. 1999, Blatt 2002, Chernomordik & Kozlov 2005, See Figure 4a), none mentions the fusion between a half-unit membrane and a cytoplasmic membrane (CM).

There are an increasing number of well-preserved plant fossils with various chemical species, cytoplasm residues and organelles preserved (Taylor & Millay 1977, Niklas 1976a,b& 1976c, 1981, Niklas & Giannasi 1977, Niklas et al. 1978, 1982, 1985, Golenberg et al. 1990, Soltis et al. 1992, Kim et al. 2004, Schönhut et al. 2004, Wang 2004,2006, 2007a&b, Collinson et al. 2005, Koller et al. 2005, Ozerov et al. 2006, Gupta et al. 2007a& 2007b, Wang & Cui 2007). Among them, Taylor & Millay (1977) used TEM (transmission electron microscope) for the first time to study the ultrastructures in cytoplasm remains of fern spores from the Pennsylvanian even though their materials did not allow them to extract too much information. Niklas et al. (1976–1985) not only extracted phytochemical species from fossil plants ranging from the Devonian to Oligocene ages, but also studied the coalified fossil materials from the Miocene in US and demonstrated the existence of chloroplasts, biomembranes, nuclei, and possible mitochondria in these fossil plants. Schönhut et al. (2004) once again found many chloroplasts in Metasequoia leaves from the Eocene in Canada and discussed their preservation mechanisms and environmental implications. Wang and his colleague (2004–2007) studied the charcoalified plant materials from the Mesozoic-Cenozoic in US and China, finding well-preserved fossil cytoplasm with ultrastructures, and proposing a new fossilizing mechanism (including lightning and wild fire). They also discussed the potential physiological value of chemical information in the cytoplasm of fossils. Gupta et al. (2007a & b) studied the preservation of cuticles from Miocene in France and Oligocene in Germany, found multiple chemical species (lignin, polysaccharide, protein and chitin) in fossil materials, and also mentioned the cellular structures preserved in epidermal cells in fossil conifers. Collinson et al. (2005) studied the taphonomy and preservation of fossil plants in Clarkia, Idaho, US, demonstrating clearly the existence of ultrastructures including chloroplast in the Miocene fossil plants. From the same locality, Golenberg et al. (1990), Soltis et al. (1992) and Kim et al. (2004) have independently extracted DNA sequences from fossil plant materials three times. Koller et al. (2005) studied a cypress fragment enclosed in Eocene amber from Russia with the organelles preserved intact. Ozerov et al. (2006) demonstrated the presence of stainable DNA in fossil plant cells in the Eocene of Russia and attributed the preservation to the presence of tannins. However, all of these works are about the static structures in plant cells, and to our best knowledge, there is no report on the physiological processes, such as membrane fusion or exocytosis, in any fossil plants yet. This paper reports membrane fusion in plant cells 15–20 Ma (million years) old. This new evidence sheds additional light on membrane fusion and the structure of the vesicle membrane, and proposes a new membrane fusion pattern hitherto unknown.

Materials and methods

One mummified coniferous cone was studied in this research. The premature cone (PB20715) was collected from the Miocene at Clarkia, Idaho, US (P33, 47°01′N, 116°25′W, Yang et al. 2005) in 2005. Originally, the cone was embedded in a gray siltstone, a sediment formed in a storm-influenced lake under anoxic conditions (Yang et al. 2005). The sediment was composed almost completely of diatom.

The fossil was recognized and photographed using a Leica MZ-16A stereomicroscope with digital camera. Tiny pieces of the organic material were sampled for microscopic observation. The fossil material was placed sequentially in 20% HCl, 40% HF, and then 20% HCl to remove inorganic minerals. In order to remove minerals completely, this processing was repeated twice more. The sample was then observed under a Leon 1530 VP SEM (scanning electron microscope) at Nanjing Institute of Geology and Palaeontology, Nanjing, China, and EDXMA (energy dispersive X-ray microanalysis) was carried out on the samples on the same SEM. Afterwards, tiny pieces of the samples were embedded in Epon 812 for thin and ultrathin sections at Nanjing Normal University, Nanjing, China. The recipe for the 20ml resin solution was 10.28 ml Epon 812, 1.24 ml DDSA, 8.48 ml MNA, and 0.34 ml DMP-30. The samples were put in acetate for 3 h, 1 h each in 50% and 67% Epon resin solutions in acetate, then a 100% resin solution overnight. Then samples were immersed in fresh pure Epon resin in a container and cured in a progressively warmer oven set at 30°C for 24 h, 45°C for 24 h, and 60°C for 24 h. Then the cured blocks were trimmed and sectioned using a Reichert-Jung Ultracut E ultramicrotome set at an interval of 2 µm using a glass knife for light microscopy and SEM (Figure 1c,d), and with a Leica Ultracut R ultramicrotome set at an interval of 70 nm using a diamond knife for TEM (Figures 2 and 3). The thin sections were photographed using an Olympus polarizing microscope (model BX51-75J21PO standard set). The ultrathin sections were stained with lead citrate. About 85 ultrathin sections in total on 12 grids were observed under Jeol JEM-1230 electron microscope at Nanjing Institute of Geology and Palaeontology, Nanjing, China. The thin sections were processed with saturated solution of NaOH in methanol alcohol to remove the embedding resin before further SEM examination for comparison (Figure 1c). The electron micrographs were saved in TIFF format, and pieced together for publication using Photoshop 7.0.

Figure 1.  A mummified coniferous cone with cytoplasm preserved from the Miocene (15–20 Ma) at Clarkia, Idaho, US. Specimen number PB20715, deposited in the Palaeobotanical Collection, Nanjing Institute of Geology and Palaeontology, Nanjing, China. (a) The fossil cone still embedded in the sediments. Note the cone axis (CA) and bract-scale complexes attached (arrows). Bar = 2 mm. (b) A cross view of a bract with the SEM. Note the intact cells and their contents (arrows) in the tissue. Bar = 20 µm. (c) A view of a bract thin section with the SEM. Note the epidermal and other cells, cytoplasm (CP) filling up the lumina, possible resin duct (RD), the cleavages between cells and their cell walls, and the ruptured cuticle (arrow). Section is about 2 µm thick. Bar = 10 µm. (d) A view of a cross-section of a bract-scale with the light microscope. Note the dark-colored cytoplasm in contrast to the light-colored cell walls in this unstained section. Bar = 0.1mm. This Figure is published with the permission of Nanjing Institute of Geology and Palaeontology, Nanjing, China and is reproduced in colour in Molecular Membrane Biology online.

Figure 2.  Vesicles and their relationships with CM in epidermal cells of a cone bract. All TEM electron micrographs. (a) A view of a cross-section of a bract. Note the faint-stained cell walls and dark-stained cytoplasm. The rectangular area is enlarged in Figure 2b. Reproduced with permission from Wang (2007b). Bar = 5 µm. (b) A detailed view of an epidermal cell, enlarged from the rectangle in Figure 2a. Note the dark-stained cytoplasm and faint-stained cell walls. The surface of the tissue is to the lower right. The rectangular region is shown in detail in Figure 2c. Bar = 1 µm. (c) A detailed view of a portion of the cell shown in the rectangle in Figure 2b. Note the relationship between the cuticle (CU), cell wall (CW), cytoplasmic membrane (CM), and cytoplasm (CP). Bar = 500 nm. (d) A detailed view of the vesicle fused to the CM, enlarged from the rectangle in Figure 2c. Note that the vesicle's half-unit membrane is connected only with the inner leaflet of the CM (arrow), which becomes wavy near the fusion pore. The outer leaflet of the CM at the fusion pore is barely visible at the fusion pore. The CM is clearly two-layered. Reproduced with permission from Wang (2007b). Bar = 100 nm. (e) A spherical vesicle with a half-unit membrane in the cytoplasm. Bar = 100 nm. (f) A vesicle connected with the CM through a cylindrical connection. Note the connection between vesicle membrane and the CM (black arrow), and the still-visible transverse membrane (white arrow) at the bottom of the vesicle. Bar = 100 nm. (g) The half-unit membrane of a vesicle connected with the inner leaflet of the CM. Note the connection between vesicle membrane and the inner leaflet of the CM (white arrow). Bar = 100 nm.

Figure 3.  Vesicles’ membranes and their relationship with double-layered cytoplasmic membrane. All TEM electron micrographs. (a) A half-unit membrane of a vesicle connected to the inner leaflet of the CM. Note the connection between vesicle membrane and the inner leaflet of the CM (white arrow) in contrast to the dangling outer leaflet of CM (black arrow). Bar = 100 nm. (b) Half-unit membranes of several vesicles connected with the inner leaflet of the CM. Note the connection between vesicle membrane and the inner leaflet of the CM (white arrows) in contrast to the outer leaflet of CM (black arrow) parallel to the inner leaflet. Bar = 100 nm. (c) A half-unit membrane of another vesicle connected with the inner leaflet of the CM (white arrow). Note the dangling outer leaflet of CM (black arrow) and the drastic variation in CM thickness on each side of the vesicle. Bar = 100 nm. (d) Half-unit membranes of a vesicle connected with the CM. Note the connection between vesicle membrane and the inner leaflet of the CM (white arrows) in contrast to the dangling outer leaflet of CM (black arrow), and two more vesicles in the cell. Bar = 100 nm. (e) Several vesicles with half-unit membranes crowded along the CM. Note that the CM is highly disturbed with its double-layered structure sometimes still visible (white arrow), and that two adjacent vesicles may have their membranes closely spaced (black arrow). Bar = 100 nm. (f) A vesicle connected with the CM. The vesicle has a unit membrane (quite different from those in Figure 3a–e, white arrow). The vesicle and the CM form an omega-shaped configuration with a narrow neck (black arrow). The vesicle membrane may become thicker and obscure due to oblique orientation of the membrane relative to the sectioning plane. Bar = 100 nm. (g) A typical double-layered structure of the CM. Bar = 100 nm. (h) A couple of the adjacent spherical vesicles before fusing with the CM. Bar = 200 nm. (i) Many vesicles with a half unit membrane in cytoplasm. Note the size and shape of the vesicles may vary from one to another. Bar = 200 nm. (j) More vesicles in the cytoplasm. Note the relationship between vesicles (arrows), and the varying membrane structure even within the same vesicle (lower arrow). Bar = 100 nm.

Results

The fossil studied is a three-dimensionally preserved mummified coniferous cone, about 5 mm long and 3.5 mm in diameter (Figure 1a). It is composed of a central axis with bracts and scales spirally arranged on the cone axis (Figure 1a). The EDXMA indicates that the elemental composition of the fossil material is mainly carbon and oxygen (C 58.77–82.15%, O 17.07–39.34%, by wt.; H is not detectable in EDXMA). The epidermal cells as well as those in the ground tissue are preserved almost intact (Figure 1b–d, 2a–d). Cytoplasm residue is found in most cells (Figure 1b–d, 2a–c). Under SEM, the cytoplasm appears as blocks of material separated by cell walls (Figure 1b–c). A similar situation is also seen under the light microscope (Figure 1d), and at this time the cells and the cell walls are distinguished by their different colors (Figure 1d). Under TEM, the cytoplasm is relatively dark-stained with lead citrate, while the cell walls are faint-stained (Figure 2a–c). Many vesicles are seen in the epidermal cells (Figure 2 & 3). A vesicle is spherical when singular in the cytoplasm and has a half-unit membrane (Figure 2e, 3h,i, 4b) or a unit membrane (Figure 3f, 4a). Vesicles may be isolated (Figure 2e–g, 3d,i) or attached each other (Figure 3e,h–j). There appears to be a cylindrical connection between a vesicle and the CM when the vesicle is about to fuse with the CM (Figure 2f, 4b). The CM typically appears as two parallel light lines, corresponding to its inner and the outer leaflets (Figure 2c–d,g, 3a–d,g, 4a,b), and about 12.5 nm (Figure 3c) to 40 nm (Figure 3f) thick, depending on their orientation and original condition (Figure 2c,d,g, 3a–d,g). The CM may become wavy or otherwise disturbed when interrupted by one or more spherical or omega-shaped vesicles (Figure 2c,d,f,g, 3a–f, 4a,b). The outer leaflet of the CM near the fusion pore either appears disturbed (Figure 2c,d,f, 3d, 4b), disappears (Figure 3a,c,d) or remains intact (Figure 2g, 3b). Drastic change in CM thickness and configuration may occur in a very limited area (Figure 3c). The vesicles of various shapes (spherical to omega-shaped) are defined by one (Figure 2c,d,e, 3a–e,h-j, 4b) or two curved lines (Figure 3f, 4a). The thickness of the double-layered vesicle membrane may range from 12 nm (Figure 3f, white arrow) to more than 40 nm (Figure 3f) in the same vesicle due to varied local orientation of membrane relative to the sectioning plane.

Discussion

Demineralizing using HF-HCl is a reliable way to remove minerals from charcoal (black carbon) that has been accepted and practiced by chemists (Poirier et al. 2000, 2002, Eusterhues et al. 2003). Therefore it is safe to assume that after three-time HF-HCl processings, only material of organic origin is left in this fossil material. It is apparent that the only source for the organic material in this fossil is plant tissues and their contents. This assumption is supported by the EDXMA analysis that indicates the composition of the fossil materials (C 58.77–82.15%, O 17.07–39.34%, by wt.) is similar to that of recent charcoal of Pinus stem (Baldock & Smernick 2002, Nguyen et al. 2004) and therefore confirms its plant origin. Clarkia, Idaho, US is a worldwide famous Lagerstätte for fossil plants (Yang et al. 2005). Niklas et al. (1985) have collected their fossil materials from this locality, extracted chemical species of phytotaxonomical values, and demonstrated the existence of chloroplasts, nuclei, biomembranes and possible mitochondria in their fossils. Recently, Collinson et al. (2005) collected their materials with superior ultrastructures, including those of chloroplasts and nuclei, from this locality. Furthermore, Golenberg et al. (1990), Soltis et al. (1992) and Kim et al. (2004) have independently and successfully extracted DNA sequences from fossil plants collected at this locality for three times. All of these works point to the superior preservation of fossil plants and the potential existence of ultrastructures in fossil cytoplasm in the cone reported here. The fact that there is no shrinkage of the cells in the cone tissues suggests that the cells are preserved almost in their life condition. Considering all of this, it is not surprising to find fine structures in this fossil cone even though physiological processes such as membrane fusion are somewhat beyond expectation.

Unlike those of recent material that has to undergo artificial fixing where artifacts may be introduced, the fossil material here does not need artificial killing or fixing and therefore eliminates the possibility of artificial errors due to killing/fixing. This is not hard to conceive because if the fossil cytoplasm was not fixed before being fossilized, we could see nothing as it would have disappeared long before considering 15–20 Ma have elapsed (Yang et al. 2005). So although we do not know exactly what kinds of natural factors were involved in the fossilization, we are not concerned with the problems of fixing and its concomitant artifacts in this case.

The material of the fossil is of charcoal, which is very stable and inert to most chemicals. This explains why charcoal preserves well (Patterson et al. 1987), and also eliminates the possibility of artifacts otherwise possibly introduced during the embedding and staining. Although it is true that the fact reported here is provocative, there are ways to test its faithfulness: (i) by independent research on other fossil materials, and (ii) by comparison between different portions of this fossil material. For the first case, whatever the future studies will indicate, now we have to be patient and wait for further works. Whoever first finds this phenomenon must make it is possible for other people to be able to test the correctness of the findings. For the second case, not all cells in the tissue demonstrate exocytosis and membrane fusion. Since the whole tissue has undergone the exact same processing, the patterns of the differentiated electron density visualized under TEM, namely the TEM micrographs, should be correlated with the original heterogeneous patterns of different constituents and/or features in the fossil material. These micrographs are the grounds for interpreting the ultrastructures in this fossil plant.

Vesicles play an essential role in communication and cargo transport in plant cells (van der Woude et al. 1971, Takeo et al. 1973, Meindl et al. 1992, Hohl et al. 1996, Thiel et al. 1998, Battey et al. 1999, Homann & Thiel 1999, Weise et al. 2000, Blatt 2002). Exocytosis occurs when the CM and the vesicle form an omega-shaped configuration, a fusion pore is formed between them, and the vesicle contents are released into the extracellular space (Thiel & Battey 1998, Battey et al. 1999, Weise et al. 2000). At this time, the cells are characterized by invagination of CM (Takeo et al. 1973, Esau 1977, Chandler & Heuser 1979, Buvat 1989). In this fossil plant material, the above dynamic process cannot be seen because the fossil materials and their electron micrographs are static. However, the electron micrographs in Figure 2c,d, f, g and 3a–f closely resemble the omega shapes of vesicles in recent plants (Figures 8–9, Takeo et al. 1973, Figure 11, Chandler & Heuser 1979, Figure 6, Chandler & Heuser 1981, Figure 2, Heuser & Reese 1981, Figure 3a, Rao & Dave 1983, Figures 1 and 4, Thiel & Battey 1998, Figure 3, Battey et al. 1999, Figure 1a, Marsh & McMahon 1999, Figure 5, Weise et al. 2000, Figure 1, Blatt 2002, Figure 1b,c, Higgins & McMahon 2002, Figure 2c,f, Stefanowska et al. 2002, Figure 4a–d, Jeremic et al. 2003, Figure 5c, Jena 2005a), thus the various shapes of many vesicles (Figure 2c,d,f,g, 3a–f) altogether imply various stages of their continuous fusing with the CM. By the way, there is no information on the origin of these vesicles even though there are other biomembranes in cytoplasm probably related to these vesicles (Supplementary figure – Online version only).

Figure 4.  Diagram showing two different membrane fusion patterns. (a) A vesicle with a unit membrane and its fusion with double-layered cytoplasmic membrane. Note that both the inner and the outer leaflets of the CM are connected with their counterparts in the vesicle membrane. (b) A vesicle with a half-unit membrane and its fusion with double-layered CM. Note that only the inner leaflet of the CM is connected with the vesicle membrane while the outer leaflet of the CM is slightly distorted due to the impact of the vesicle content. This is a new pattern proposed based on the observation on cells in this fossil material.

Normally, as in recent plant cells, the CM appears as two smooth parallel lines around cytoplasm (Figure 2c,d,g, Figure 3a–c,g, Figure 4a,b). Most membrane fusions in the cone occur between a vesicle's half-unit membrane and CM's inner leaflet, while the corresponding outer leaflet of the CM either remains intact (Figure 2g, Figure 3b, leftmost white arrow), or appears disturbed (Figure 2c,d, Figure 3d). The disturbed outer leaflet suggests that it is under pressure or impact from the vesicle contents. The vesicles and their contents appear to cause great changes in CM profile and thickness (Figure 2d, Figure 3a–f). The CM may be as thin as 12.5 nm (Figure 3c), very close to that in recent materials (9–11 nm, Morré 1990), while it could be up to 40 nm thick when highly disturbed (Figure 3f). This variation scope appears wide at the first glance; however, a similar drastic variation has been reported for CM, ranging from 5 nm up to 18 nm, in recent cells (Korn 1966). Considering that the CMs in this fossil material are highly disturbed and that those in recent materials (Korn 1966) are just in resting status, the drastic variation in CM thickness appears less surprising. And most of the thicknesses of the CMs in this fossil fall in between the two extremities (Figure 2d,g, Figure 3a–e,g), depending on orientation and other factors (Korn 1966). The drastic variation in CM thickness from 12.5 nm to 31 nm within 300 nm (the left and right of the fused vesicle in Figure 3c) implies that the increased thickness of the CM is due more to the original membrane disruption rather than an oblique sectioning and that the image (Figure 3c) more or less reflects the true status of the CM even though the counterpart of such a drastic variation has not been seen in recent cells yet.

Membrane fusion is a process ubiquitous and fundamental to eukaryotic cells (Gruner 2002, Yang & Huang 2002, Jahn et al. 2003, Tamm et al. 2003, Ostrowski et al. 2004, Koumandou et al. 2007, Wong et al. 2007), and it is closely related to the structures of the membranes involved in. Although vesicle trafficking has been intensively studied, little is known about the membrane of secretory vesicles (Battey et al. 1999). On one hand, it is believed that vesicle membranes have a structure similar to that of the CM (Buvat 1989), and it is generalized that all biological membranes are unit membranes with a double-layered structures (Holum 1978, Staehelin & Newcomb 2002). Thus it is not surprising that almost all of the models of membrane fusion concern fusion between two unit membranes (Palade & Bruns 1968, Lucy 1970, Poole et al. 1970, Thiel & Battey 1998, Blatt et al. 1999, Blatt 2002, Tamm et al. 2003, Tuma & Hubbard 2003, Jena 2005a&b, Chernomordik & Kozlov 2005, Wong et al. 2007, Figure 4a). On the other hand, although half-unit membranes, composed of a monomolecular layer of radially-oriented phospholipid elements (Hodge et al. 1956, Csillik 1963, Cardell et al. 1967, Ambrose & Easty 1979, Hilhorst et al. 1982, Scherz et al. 1990), have been intensively studied by biochemists (Fromherz & Rüppel 1985, Moscho et al. 1996, Oliveira et al. 1998, 2005, Brockman 1999, Deleu et al. 2005, Rosetti et al. 2005), in living cells they only rarely are reported (for example, mouse liver and muscle, mature cells and young apical cells of Nitella cristata, root tips of germinating wheat, and leaves of Zea mays, Hodge et al. 1956; Rana esculenta and Mus rattus, Csillik 1963; rat, Cardell et al. 1967; Triticum aestivum, Anderson et al 1970; Allium cepa, Brassica capitata, Gossypium hirsutum, Yatsu et al. 1971; Ranunculus sceleratus, Konar et al. 1972; Arachis hypogaea, Yatsu & Jacks 1972; Tilletia caries, Gardner & Hess 1977; Desulfovibrio vulgaris, Hilhorst et al. 1982; Pinus pinea, de Castro & Martinez-Honduvilla 1984; Rhodospirillum rubrum, Scherz et al. 1990; Zea mays, Somerville et al. 2002; Arabidopsis, Schumann et al. 2003). It is believed that a half-unit membrane provides a good package for storage and transport of photoproducts, enzymes (Hodge et al. 1956, Csillik 1963, Cardell et al. 1967, Yatsu et al. 1971, Yatsu & Jacks 1972, Hilhorst et al. 1982, Scherz et al. 1990, Somerville et al. 2002), and hydrophobic drugs (Savić et al. 2006). Among them, Anderson et al. (1970, Figure 1c) and Yatsu et al. (1971, Figure 6a) demonstrate clearly a half-unit membrane in contrast to a unit membrane in the same pictures. However, despite these reports of half-unit membranes, they have been ignored in membrane fusion research and none of the membrane fusion models mention fusion with a half-unit membrane.

In this research, transcytoses (probably all exocytoses) observed in the fossil cone consistently demonstrate fusions between a half-unit membrane of a vesicle and a unit membrane of CM (except in Figure 3f). Many of the vesicles in the epidermal cells of the cone have a single-lined profile and fuse only with the inner leaflet of the CM (Figure 2c,g, Figure 3a–e, Figure 4b). This strongly suggests the existence of half-unit membranes and a membrane fusion pattern ignored hitherto in plant cells. An unfavored interpretation for this phenomenon is that the single-lined profile of the vesicle actually stands for a unit membrane, thus there is no hassle of the so-called half-unit membrane. However, it follows that each cell has two cytoplasmic membranes around since two parallel lines are seen around the cytoplasm (Figure 2c,g, Figure 3a–e) and that the vesicle in Figure 3f also has two unit-membranes around. Apparently, these deductions, especially the first one, are hard to accept for most botanists. However, if people adopted this idea, a plastid, nucleus or mitochondrion (the only organelles with two unit membranes around) transferred across the CM would be a phenomenon of great scientific importance. Due to the difficulty to accept two cytoplasmic membranes around a single cells and the non-existence of a plastid/nucleus/mitochondrion transferred across the CM, this alternative is discarded hereafter. On the contrary, the half-unit membrane interpretation is in good agreement with experimental results, in which vesicles can be formed on a lipid monolayer (Higgins & McMahon 2002). The CM's biochemical properties and the way it fuses with a vesicle membrane (Figure 2c,d,g, Figure 3a–e) suggest that the hydrophilic ends of the phospholipid elements on the vesicle membrane point to the exterior of the vesicle, and the hydrophobic ends to the interior. This type of membrane appears convenient for membrane fusion, vesicle membrane recycling, transport of hydrophobic product, and also is ideal for the kiss-and-run type of exocytosis in plant cells since there is no need to interact with the outer leaflet of the CM, in which the process is otherwise envisaged as a difficulty by modern biologists (Higgins & McMahon 2002). Interestingly, in the same plant (even possibly in the same cell) there are fusions between two unit membranes (Figure 3f) as well as between a half-unit membrane and a unit membrane, and both types of membranes are distinct under the TEM (Figure 2c,d,f,g, Figure 3a–f). This implies that two membrane fusion patterns truthfully exist simultaneously even though one of them has been overlooked or unsubstantiated for a long time, and that the contents in these two kinds of vesicles should be of different biochemical properties.

A unit membrane of the vesicle appears as a light-colored curved belt of more or less uniform width (Figure 3f), and its double-layered structure may still be visible sometimes (Figure 3f white arrow). The thinner portion of the double-layered vesicle membrane is only 12 nm thick (Figure 3f, arrow), while the thicker one in Figure 3f is up to 40 nm thick (probably due to the local oblique orientation). This drastic change is understandable since the local orientation of the vesicle membrane, which is close to spherical in form, relative to the sectioning plane may vary from point to point.

Interestingly, the good preservation of this fossil plant allows the transient membrane-fusion intermediate structure (Chernomordik & Kozlov 2005, Wong et al. 2007) to be observed herein (Figure 2f). It appears that there is a cylindrical connection established between the half-unit membrane of the vesicle and the CM before they fuse (Figure 2f, black arrow) while the vesicle has not completely lost its spherical form (Figure 2f, white arrow). This is in good agreement with models of membrane fusion (Blatt et al. 1999, Blatt 2002, Gruner 2002, Yang & Huang 2002, Jahn et al. 2003, Tamm et al. 2003, Tuma & Hubbard 2003, Jena 2005a&b, Chernomordik & Kozlov 2005, Wong et al. 2007) even though the actual structure has never before been directly observed in recent cells under TEM.

As more advanced technologies become available for application recently, more and more subcellular secrets of fossil plants are revealed. Schönhut et al. (2004) have revealed the well-preserved chloroplasts in cells of Eocene Metasequoia leaves from Canada, and Ozerov et al. (2006) have observed stainable DNA in cells of Eocene plants from Russia. Both of the groups agree with Niklas et al. (1978, 1985) on the preservation mechanism: tannins preserve the subcellular structures. Koller et al. (2005) add another alternative for preservation by pointing out the preservation potential by amber for cellular details in an Eocene plant from Russia. It is important to note that the ultrastructures observed in Schönhut et al. and Koller et al.'s materials are especially similar to those in this paper, in terms of membrane structures and negative-staining-like appearance. In addition, Wang & Cui (2007) and Wang (2004, 2006, 2007a) & b) have proposed that some natural phenomena such as lightning, wild fire and high temperature may contribute to the exceptional preservation of ultrastructures in fossil cytoplasm. In spite of so many alternatives for preservation of cellular details, it remains true that little is known of the preservation mechanism of the fine ultrastructures in this fossil. However, this only means that people do not understand it much, and does not mean that the preservation mechanism does not exist. It does not mean that the unknown mechanism does not work, either. Similar situation exists in biological study on recent material. Microwave fixation is a good way to fix biological samples but its mechanism is not completely understood (Login & Dvorak 1994). However, this not-understanding does not stop people from using it in biological and medical research. Actually, all ‘correct’ artificial fixing and processing must have more or less changed the original status in plant cells. It is also right to assume that changes have occurred in fossil cytoplasm. Since the quality of the images in this fossil material are comparable to their recent counterparts and those in fossil plants (Niklas et al. 1978, Schönhut et al. 2004, Koller et al. 2005), it is not a bad idea to accept their existence for the time being and then try to figure out the mechanism later. The information in this paper implies that that there might be unknown bypasses for biological sample preparation. This challenges scientists working on recent materials and provides an initiative for the further understanding of nature, which is the only and reliable source of our knowledge.

The presence of an unknown membrane fusion pattern in this fossil plant has two possible mutually exclusive implications. (i) It is a pattern only seen in fossil plants: therefore its presence in this Miocene plant and absence in recent plants suggest that plants have dropped this pattern during evolution. If so, this would be the first time that we have understood plant evolution at this physiological level. (ii) This membrane fusion pattern also exists in recent plants but people simply have not observed it in recent plants yet. We favor the latter alternative because (i) the age of the fossil is 15–20 Ma, which appears too short for such a drastic variation to occur; and (ii) there have been, though rare, reports of half-unit membranes in recent plants (Hodge et al. 1956, Csillik 1963, Cardell et al. 1967, Anderson et al 1970, Yatsu et al. 1971, Konar et al. 1972, Yatsu & Jacks 1972, Gardner & Hess 1977, Hilhorst et al. 1982, de Castro & Martinez-Honduvilla 1984, Scherz et al. 1990, Somerville et al. 2002, Schumann et al. 2003) and some of them contain secretory contents, and thus they (half-unit membranes) have to fuse with CM (unit membranes) somehow somewhere sooner or later. Theoretically, this type of membrane fusion is inevitable for plant cells. It appears not impossible for scientists to find this type of membrane fusion in recent plants some time later.

Supplementary Material

Acknowledgements

We thank Dr Zhiyan Zhou for his support during this research, Drs Jinxing Lin, Chuanming Zhou, Yongdong Wang, Yi Wang, Yuandong Zhang, Ms Chunzhao Wang, Mr Yongqiang Mao, Mr Erjun Zhuo, Ms. Cuiling He, Ms Fangming Xu, Drs William Schopf, Margaret Collinson, Gaetan Guignard and Johanna Van Konijnenburg-Van Cittert for their help during this research, Dr Douglas McKinnon and Ms Margaret Joyner for help on English, two anonymous reviewers for help with the manuscript. This research is supported by Jiangsu Planned Project for Postdoctoral Research Funds, China Postdoctoral Science Foundation (No.2005037746), K. C. Wong Post-doctoral Fellowships, State Key Laboratory of Palaeobiology and Stratigraphy (Nanjing Institute of Geology and Palaeontology, CAS), National Natural Science Foundation of China Programs (No. 40632010 and No.J0630967). This paper is a contribution to IGCP 506.