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Journal of Science 113, 81-89 (2000) 81 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0929

Plastid tubules of higher are -specific and developmentally regulated

Rainer H. Köhler* and Maureen R. Hanson Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca NY 14853, USA *Author for correspondence (e-mail: [email protected])

Accepted 28 October; published on WWW 9 December 1999

SUMMARY

Green fluorescent stroma filled tubules (stromules) particular developmental zones of the . Furthermore, emanating from the surface were observed in suspension cells of tobacco exhibit stromules on virtually transgenic plants containing plastid-localized green every plastid with two major forms of appearance. The fluorescent (GFP). These transgenic tobacco plants majority of cells show a novel striking ‘octopus- or were further investigated by epifluorescence and confocal millipede-like’ structure with plastid bodies clustered laser scanning microscopy (CSLM) to identify around the nucleus and with long thin stromules of up to developmental and/or cell type specific differences in the at least 40 µm length stretching into distant areas of the abundance and appearance of stromules and of . cell. The remaining cells have plastid bodies distributed Stromules are rarely seen on -containing throughout the cell with short stromules. Photobleaching plastids in cell types such as , guard cells or experiments indicated that GFP can flow through mesophyll cells of . In contrast, they are abundant in stromules and that the technique can be used to distinguish tissues that contain chlorophyll-free plastids, such as interconnected plastids from independent plastids. and root. The morphology of plastids in and is highly dynamic, and plastids are often elongated and Key words: Plastid, Stromule, GFP, Photobleaching, Transgenic irregular. The shapes, size, and position of plastids vary in

INTRODUCTION on the investigation of fixed material by electron microscopy (EM). EM provides very high resolution but fixation artifacts , because of their role in transforming the energy and poor preservation of structures might result in of sunlight into chemical bonds for fixation of carbon, have misinterpretation or failure to detect sensitive structures. been the most intensively studied type of plastid. However, Furthermore, fixing the tissue for EM studies prevents non-chlorophyll containing plastids, though not involved in examination of dynamic processes; these can be seen only by carbon fixation, play key roles in other biochemical pathways labelling of and subcellular structures in living cells. in the plant. Colorless non-chlorophyll containing plastids, In order to make all plastid types accessible to observations often called , are found in a wide variety of tissues in vivo we labeled plastids with green fluorescent protein and cell types such as roots, petals or suspension cells (Kirk (GFP). GFP is a novel marker protein derived from the and Tilney-Bassett, 1978). Specialized leucoplasts can be jellyfish, Aequorea victoria, that emits green fluorescent light distinguished by the compounds stored as energy reserves. when excited with blue light (Chalfie et al., 1994). Expression storing plastids that are common in roots and some of GFP fused in frame with the recA-transit peptide (CT-GFP, suspension cells are called , protein storing plastids Cerutti et al., 1992) regulated by a constitutive 35S CaMV are , storing plastids are and promoter in transgenic plants permits observation of plastids carotinoid-containing plastids, present in some roots, and by confocal laser scanning microscopy or epifluorescence flowers, are called . Most plastid types as well as microscopy in vivo (Köhler, 1998). The CT-GFP fusion protein proplastids are thought to be able to differentiate into each as well as CT-GFP fluorescence are specifically associated with other, with exceptions such as , plastids in the soluble stroma fraction of purified plastids and are not senescing leaves, which are believed to be a non-reversible detectable in a membrane fraction that includes broken and terminal differentiation (Strasburger et al., 1991). intact membranes. Green fluorescent tubular Non-chlorophyll containing plastids have been more structures can be observed emanating from the chloroplasts in difficult to observe by light microscopy than chloroplasts, CT-GFP-expressing transgenic plants (Köhler et al., 1997). restricting their characterization in vivo. Current information Because the imported GFP is localized to the stroma, according about non-green plastid shapes and distribution is mostly based to fractionation experiments, we named these tubules 82 R. H. Köhler and M. R. Hanson stromules, for stroma filled tubules. Stromules are highly channels of Adobe Photoshop and merged into the final image. Black dynamic, variable in length and shape, and they can and white CLSM images show the green channel only because red interconnect different plastids. Photobleaching experiments fluorescence was absent. Simultaneous recording of GFP fluorescence have shown that stromules permit the direct exchange of GFP and light microscopic images was done with a BHS filter cube between separate plastids providing a novel pathway of (488DF10, Dichroic 510, emission 515LP) and fiber optics. Image communication between plastids that circumvents passage stacks taken along the optical z-axis in steps indicated in the figures were opened in Confocal Assistant 4.02 (Todd Clark Brelje, Bio-Rad, through the selective membranes or the selective import and Hercules, CA) and projected into a single plane using maximum pixel export machinery (Köhler et al., 1997). Stromules can be seen value. For light micrographs, single images were selected from the on plastids of non-transgenic spinach and tobacco plants by stacks that provided the best representation of the general appearance light microscopy (Spencer and Wildman, 1962; Wildman et al., of the cell including structures such as the nucleus, cytosolic strands 1962; Wildman, 1967; Köhler et al., 1997). and, if visible, plastid tubules. While stromules were observed in tissue of tobacco, petunia and Arabidopsis (Köhler et al., 1997), most leaf cells Photobleaching experiments lack detectable stromules, making investigations of the nature The areas indicated as boxes in Fig. 5A and 7A were photobleached and function of this novel plastid structure cumbersome. By by repeated scanning at full power with a 15 mW krypton/argon mixed fluorescence microscopy we examined the appearance of gas laser using the K1/K2 filter set. The laser was restricted to this area by using the zoom function of the COMOS program. A Zeiss plastids and abundance of stromules in a number of different Plan Apochromat ×63 oil immersion objective with NA 1.4 was used tissue types in transgenic tobacco plants expressing GFP for photobleaching. Images before and after photobleaching were localized to plastids. Our results indicate that plastid collected with only 10% laser power to reduce photobleaching and appearance and stromule number is highly dynamic and possible damage to the cells. variable between different tissues and cells, with the most extensive and numerous stromules present in cells containing non-green plastids. RESULTS Abundance and appearance of plastids and MATERIALS AND METHODS stromules in different plant tissues Expression of CT-GFP targeted to the stroma of plastids does construct not result in any visible phenotype in the transgenic plants. The modified GFP4 coding region (Haseloff et al., 1997), which was Transgenic plants and suspension cultures grow normally, the further altered by a serine to threonine at codon 65 plants have normal set, and the transgenes segregate as (S65TmGFP4), was fused in frame with the Arabidopsis recA transit expected. In order to identify variations in the abundance of peptide (CT-GFP; Cerruti et al., 1992). This construct was cloned into stromules and in the appearance of plastids in different tissues, a plant expression vector containing the double 35S CaMV promoter we investigated tobacco plants by epifluorescence microscopy (Datla et al., 1993; Tobias and Nasrallah, 1996) and the transformation initially (data not shown) and then used confocal laser scanning of tobacco Petit Havana was done as described by Horsch et al. microscopy for imaging. The frequency of stromules in (1985). chlorophyll-containing cells is very low (Fig. 1A,B,C). Most Growth of plant material chlorophyll-containing cells of a given cell type do not exhibit Leaf and root tissue were obtained from transgenic tobacco plants stromules. Nevertheless, stromules are observable in all of (Nicotiana tabacum c.v. Petit Havana) grown in the green house in the cell types examined, such as guard cells, trichomes, soil or under sterile conditions in Magenta boxes on N-13 medium parenchyma cells, mesophyll cells and epidermal cells. Even (Hosticka and Hanson, 1984) at 25¡C under a 16 hour light, 8 hour in cells that have chloroplasts with visible stromules extending dark cycle. There was no difference in plastid appearance between from their surfaces, other chloroplasts in the same cell lack plants grown in soil or in vitro. Callus growth was induced by stromules (Fig. 1A,B,C). Often a single plastid with stromules incubation of sterile leaf material from plants kept in Magenta boxes is visible in a cell surrounded by other chloroplasts in the same on NT1 agar medium (MS basal salt mixture (Sigma M 5524, St cell and surrounded by other cells that do not exhibit detectable Louis, MO), 100 mg/l myo-inositol, 1 mg/l thiamine, 0.2 mg/l 2,4-D, stromules. Due to the very low background fluorescence, bright 180 mg/l KH2PO4, 30% sucrose, and 9 g/l Phytagar at a pH of 5.8). Suspension cells were grown from callus in liquid NT1 medium and green fluorescent stromules can be found against a virtually transferred every week to fresh NT1 medium. Cultures were grown at black background even when present on a single in 110 rpm under a 16 hour light and 8 hour dark cycle at 25¡C. one out of hundreds of cells. Mature chloroplasts in vascular plants are generally lens- Imaging of plant material shaped structures of 5-7 µm length and 1-3 µm width and are Material from plants grown in the greenhouse or in Magenta boxes bi-convex, plano-convex or concavo-convex in profile (Hoober, was mounted on slides in 20 mM MES pH 5.5, 1 mM CaCl2. 1984; Thomson, 1974). Red chlorophyll autofluorescence that Suspension cells and callus cells were mounted in NT1 medium (s.a.). outlines the distribution of thylakoid membranes and stacks in Suspension cells were imaged 3 to 4 days after transfer to fresh NT1 chloroplasts appears as a regular shaped round or oval structure medium while they are still in logarithmic growth phase. Imaging was in chloroplasts in transgenic tobacco plants expressing GFP performed using a Bio-Rad MRC-600/Zeiss Axiovert 10 CLSM and the COMOS program (Bio-Rad, Hercules, CA). Green GFP (Fig. 1A,B,C). If single stroma enter stromules, they fluorescence and red chlorophyll autofluorescence were collected are not sufficiently autofluorescent to be detected. Imaging simultaneously with a standard K1/K2 filter set (488/568 dual band stroma-targeted GFP reveals that chloroplasts have a more pass, Dichroic 560 DCLP, 522DF35 and 585 EFLP emission filter). irregular and dynamic shape than evident from chlorophyll The separate images were imported into the red and green RGB autofluorescence imaging (Fig. 1A,B,C; Köhler et al., 1997). Plastid and stromule morphology 83

Fig. 1. Tissue-specific variations in stromule abundance and plastid appearance. CLSM images of different tobacco cell types and tissues expressing plastid targeted CT-GFP (Köhler et al., 1997). The z-axis distance between images of a stack and the number of images projected into a single plane is shown in brackets. Bars, 10 µm. Thin arrows indicate stromules and arrowheads indicate plastid bodies. Images (Ag), (Bg), and (Cg) show GFP fluorescence (green channel), images (Ar), (Br), and (Cr) red chlorophyll autofluorescence (red channel), and images (Am), (Bm), and (Cm) merged images of the green and red channels. (A) Leaf mesophyll cells (0.2 µm/28 images); (B) A cell of leaf (0.2 µm/46 images); (C) Guard cells of leaf (0.2 µm/36 images); (D) Epidermal and guard cells in the limb of a petal 5 cm in size (1 µm/13 images), red fluorescence has been enhanced relative to GFP fluorescence to make the autofluorescence visible; (E) cell of the root hair zone (0.5 µm/13 images).

Green fluorescent tubules, lobes, knobs, and loops that enclose non- fluorescent areas or particles are visible. Time-lapse studies show that in some cells these structures are amoeboid and highly dynamic, moving, changing their shape plastids in root cells (see below), chlorophyll-free leucoplasts constantly and producing tubules that extend from the plastid in corolla cells, such as hair cells and epidermal cells, are surface, move around or retract (Köhler et al., 1997; see irregular and variable in shape and size (Fig. 1D). In contrast, http://www.mbg.cornell.edu/kohler/kohler.html for movies). chlorophyll-containing plastids in guard cells of the corolla are Cells from non-chloroplast containing tissues such as petals oval or round and regular in shape. No major difference in the and roots exhibit mostly irregularly-shaped plastids with wide appearance of leucoplasts within the white or pink corolla parts variation in shape and length and differences in the abundance at different developmental stages were detected (data not and length of stromules (Fig. 1D,E). Because of this shown). The plastid bodies in the corolla, which are up to 4-5 remarkable variation in shape, it is more difficult to distinguish µm in diameter, are smaller than chloroplasts in most leaf cells between the plastid body and stromule in non-green cells than but larger than plastid bodies of root cells. Tubular plastids are in chloroplast-containing cells. The name stromule is used for more frequent in the corolla than in roots and seem to be structures that appear less than 0.8 µm in diameter; larger parts present in every cell. Corolla plastids have non-fluorescent of the plastid will be referred to as plastid body (Figs 1D,E, inclusions with diameters of up to at least 2.2 µm. 3B,C, 4G). Diameters are estimated from measurements of the fluorescent area of micrographs taken by CLSM, which usually Developmental changes in plastid appearance and results in an overestimation of the actual size. Both stromules stromules of roots and plastid bodies show green fluorescence as a result of GFP The root was selected for more detailed examination of localized in the stroma. Sometimes stromules can be seen that changes in plastid and stromule appearance because root appear not be connected to a plastid body at all (data not development is well defined and cell lineages can be easily shown). identified (Dolan et al., 1993). For descriptive purposes, the The abundance and appearance of plastids and stromules root can be divided into different overlapping zones, the were examined in floral tissue. Tobacco flower development meristematic zone, the elongation zone, the differentiation has been divided into 20 stages on the basis of size and zone, and the mature root (Benfey and Schiefelbein, 1994; morphological criteria (Drews et al., 1992). Mature stage 12 Dolan et al., 1993). Root plastids differ in their appearance, open tobacco flowers are about 5 cm in size and have a corolla length of stromules and intra-cellular distribution depending on made of five fused petals. Unusual morphology of GFP-labeled the stage of development, which is defined by their position plastids was visible in the white stalk of the corolla as well as within the root (Fig. 2). Most plastids in root cells are smaller in the pink limbs. Epidermal cells of stage 12 corolla contain than chloroplasts with a plastid body of about 1.8 to 3 µm in colorless plastids or leucoplasts and show faint red diameter and stromules of less than 0.6 µm in diameter. autofluorescence in the in the limb cells, which might Plastids and stromules in the small cells of the meristematic be a result of anthocyanin accumulation (Fig. 1D). Similar to zone are densely packed and difficult to separate even by 84 R. H. Köhler and M. R. Hanson

CLSM (Fig. 2A and B). In most cells of the meristematic zone, plastids appear organized in one big circle around a non- fluorescent area that might be the nucleus, with stromules stretching outward (Fig. 2B). In the elongation zone, stromules are longer and plastids become less densely packed but are still often around the nucleus (Fig. 2C). In the differentiation zone, plastids show elongated stromules, plastid density drops and plastids are occasionally clustered around the nucleus (Fig. 2D). In mature root cells, with increasing distance from the root tip, stromules appear shorter again and plastids become further dispersed within the cell with frequent clustering around the nucleus Fig. 2. Changes in plastid appearance during root (Fig. 2E). Stromules are development. CLSM images of tobacco root cells. visible in most root cells. The z-axis distance between images of each stack is 0.5 µm. Root hairs (h), nuclei (n) and stromules Root plastids contain small µ non-fluorescent inclusions of (arrows) are indicated. Bars, 10 m. The number of µ images projected into a single plane is indicated in less than 1 m in diameter brackets. (A) Overview of the root tip zone (42 that might be starch grains or images); (B) Meristematic zone (6 images); (C) plastoglobuli (Fig. 1E). Elongation zone (17 images); (D) Differentiation zone (27 images); (E) Mature root near the root tip Stromules in callus and (29 images). suspension cells The higher abundance of stromules and the difference in appearance of plastids in chlorophyll-free cell types, such as likely to be proplastids, which are about 1 µm in diameter and petals and roots, suggests that these differences might be do not show red chlorophyll autofluorescence. In contrast, related to plastid functions other than . To plastids in dividing callus cells are dynamic in shape and investigate if these differences can be regulated by a change of slowly lose all their chlorophyll, observable by the loss of red growth conditions that facilitates loss of chlorophyll and autofluorescence (Fig. 3B and C). The majority of these photosynthetic capacity, we initiated callus and suspension plastids consist of an enlarged portion, the plastid body, and a cultures. Chlorophyll content and the transition from tubular component extending from the enlarged portion. Both chloroplast to or is regulated by sucrose parts are highly variable and dynamic. In some cells concentration and phytohormone composition of the growth chlorophyll autofluorescence has vanished completely and medium (Kaul and Sabharwal, 1971; Sakai et al., 1996). Callus most plastids have an elongated shape with enlargements in growth was induced by placing leaves of transgenic and control one or more places. Non-fluorescent inclusions that show wide plants on NT1 medium. The phytohormone composition of the variability between cells might be starch grains (amyloplasts, NT1 medium in combination with high sucrose levels results Fig. 3B). Often several plastid bodies are connected to form in a change from autotrophic to heterotrophic growth. Red small branched networks of plastid bodies and stromules (Fig. autofluorescent chloroplasts are transformed to leucoplasts or 3C). It is not evident whether these networks originate from a amyloplasts that lack red autofluorescence. Initially, leaf cell single plastid or are fusions of originally separate plastids. chloroplasts exhibit only a few stromules and chloroplasts are Placing callus cells into liquid NT1 suspension culture oval, about 7 µm wide and 1-2 µm deep (data not shown). After promotes further striking changes in plastid structure and about four weeks on NTI medium, cells vary in their intracellular distribution. A minor fraction of the plastids in appearance. Plastids from non-dividing areas of the leaf show suspension cells exhibit structures comparable to that of callus reduced chlorophyll content and some short stromules but lack cells. They have elongated plastids with short stromules that the fluidity of shape seen in non-green cells. (Fig. 3A). Non- are distributed throughout the cell and sometimes contain non- dividing cells contain small round green florescent particles, fluorescent inclusions, likely starch grains (Fig. 4C,D,E,F). In Plastid and stromule morphology 85 contrast, the majority of cells shows striking ‘octopus-’ or extend from the plastid bodies into distant areas of suspension ‘millipede-like’ plastid structures (Fig. 4A,B,G). Most of the cells. Stromules that are close by but are obviously not plastid bodies in these cells are clustered around the nucleus connected do not change in fluorescence (Fig. 7 stromule b). and often seem to be virtually absent from other parts of the Some stromules appear to be connected, but there is no flow cell. From some plastid bodies, long stromules of up to at least of GFP between bleached and unbleached regions (Fig. 7, 40 µm reach into distant areas of the cell (Fig. 4G). Stromules stromule c). It is likely that these stromules, though in close in suspension cells are variable in shape, can be branched or proximity, are actually not fused to one another, preventing even encircle larger areas (Fig. 6A). They are highly dynamic, exchange of their contents. producing new branches, frequently retracting branches or moving about (data not shown; movies available at http://www.mbg.cornell.edu/kohler/kohler_JCS.html). DISCUSSION

Movement of GFP through stromules Long protuberances extending from chloroplasts Mitochondria of yeast cells are present in the form of a single in living cells have been reported in the literature a number of extended network during certain stages of development times (e.g. Heitz, 1936; Wildman et al., 1962; Köhler et al., (Hermann and Shaw, 1998). Although a single network of 1997). The most impressive studies prior to GFP technology plastids has not been reported in vascular plants, it is known were movies of phase-contrast images of hand-sectioned live that plant organelles at some stages of development fuse to leaf cells performed by Wildman and his colleagues (Spencer larger units. For example, mitochondria of tobacco protoplasts and Wildman, 1962; Wildman et al., 1962; Wildman, 1967). can fuse to long vermiform and branched structures (Stickens Unfortunately, these studies have largely been forgotten or and Verbelen, 1996; R. H. Köhler, unpublished observations) ignored. There are several reports of tubular membrane bridges and plastids of vascular plants and Acetabularia can be joined connecting chloroplasts in Acetabularia (Menzel, 1994) and, by tubular connections (Wildman et al., 1962; Menzel, 1994; during a particular state of the cell cycle, in Euglena (Ehara et Köhler et al., 1997). In order to investigate whether the plastid al., 1990), but these interconnections have been largely been bodies that are concentrated around the nucleus in suspension considered to be unusual traits of these unicellular cells are connected with each other to form a single extended rather than general features of chloroplasts. When stromules network, photobleaching experiments were performed (Fig. 5). were observed occasionally in vascular plants, they were Plastids close to the nucleus and parts of the stromules that usually dismissed as artifacts or abnormalities resulting from originate from them were photobleached by repeated scanning viral infection, chloroplast breakdown, or cell death. with the laser (Fig. 5A). Images taken after photobleaching Now that transgenic plants can be produced with GFP over a period of 12 minutes do not show any major flow of localized in plastids, many plastid types can be observed in fluorescent GFP from the unbleached plastids into the bleached vivo and stromules can readily be detected. It is unlikely that plastids (Fig. 5B and C) indicating that the plastids around the plastid localization of GFP causes any significant disturbance nucleus do not form a single communicating network. The of plastid morphology. The transgenic plants grow normally sensitivity and resolution of this experiment is not sufficient to and our images of plastids in transgenic plants are consistent exclude that some of the plastids might be connected and could with observations made by us and others on plastids of non- exchange GFP through stromules. Exposure to the intense laser transgenic plants. Observations of wild-type plastids in situ light during photobleaching does not damage the cells. were previously limited to naturally colored plastids such as Bleached cells show unaltered movement of stromules within chloroplasts or chromoplasts, or required electron microscopic the bleached and unbleached area as observable by comparing examination of fixed tissue. Green leaf tissue, widely used for Fig. 5A and B. The movements continue for at least several chloroplast studies, exhibits a very low frequency of stromules, hours after bleaching (data not shown). perhaps accounting for the rarity of their detection by light The independence of most plastids is also supported by the microscopy of wild-type plants. Furthermore, stromules lack observations of single images taken along the optical z-axis. detectable chlorophyll, making visualization by chlorophyll Single CLSM images that were collected by taking images autofluorescence impossible. With plastid-localized GFP, the about 0.5 µm apart from each other along the optical z-axis only limitation on tissue type that can be examined is the level indicate that most plastids around the nucleus are separate units of GFP expression that can be obtained with the gene and are not connected through fluorescent tubules (Fig. 6). regulatory sequences used to express the chimeric gfp gene and Plastids in root cells are sometimes connected by stromules, the depth of penetration possible by the microscope. The and previously we demonstrated that GFP can move through double 35S CaMV promoter (Datla et al., 1993), a highly active the root stromules from one plastid body to another (Köhler et promoter expressed in many, but not all tissues, was utilized in al., 1997). To investigate whether GFP can move freely through our studies. GFP expression was too low to observe plastids in the extremely long stromules extending from plastids near the sporogenous tissue, the , or the inner cell layers nucleus into distant areas of suspension cells, additional of the stem, but plastids in most other cell types and tissues photobleaching experiments were performed (Fig. 7). could be observed. Because stromules are often very thin, a Photobleaching of a part of a stromule by scanning a box higher level of GFP expression is needed to observe them than shaped area repeatedly with the laser led to the loss of GFP to observe GFP-labelled plastid bodies. Thus it is possible that fluorescence in the exposed area and in areas of the connected some stromules may have gone undetected even in our stromule that are not exposed to the laser (Fig. 7, stromule a). transgenic plants. Thus, there is an exchange and movement of unbleached and Because of the highly dynamic nature of the plastid envelope bleached GFP molecules through the tubular structures that and protuberances, light and fluorescence microscopy of living 86 R. H. Köhler and M. R. Hanson

Fig. 3. Regulation of stromule abundance and of plastid morphology by growth conditions. Callus cells were imaged by CLSM four weeks after transgenic tobacco leaf explants were placed on NTI agarose medium under 16 hour light/ 8 hour dark cycle for induction of callus growth. Yellow and orange color result from a combination of red chlorophyll autofluorescence and green GFP fluorescence; change from yellow or orange to green indicates decreasing chlorophyll. Bars, 10 µm. Images were taken at 1 µm intervals along the optical z- axis. The number of images projected into a single plane is indicated in brackets. (A) Non-dividing cell from the leaf area (25 images); (B) Cell from pale green callus (14 images); (C) Cell from white chlorophyll-less callus (12 images). Large arrowheads, plastid bodies; small arrowheads, proplastids; arrows, stromules.

wild-type and transgenic cells has previously demonstrated their existence most convincingly. However, some electron microscopic studies of plastids have captured images of plastid protuberances, often reported in papers describing plants stressed in one way or another (e.g. Shalla, 1962; Newcomb, 1967; Nichols et al., 1967), as the accepted paradigm for normal plants did not include the existence of tubular plastid extensions. Though some of the previously reports of extensions may be artifact, a number of the images appear to be high quality and are consistent with our observations at the light microscopy level. Furthermore, chloroplast protuberance in rice leaves were observed when Bourett et al. (1999) preserved cells by high-pressure freezing and freeze substitution. Two factors have probably contributed to the paucity of electron micrographs of stromules. First, most investigators have utilized leaf tissue, in which these structures are not common. Second, stromules are not well preserved by standard fixation methods for electron microscopy (S. Wilson and M. R. Hanson, unpublished data).

Fig. 4. Pleomorphic plastids in transgenic tobacco suspension cells. (A,C,E) Single light microscopic images of suspension cells expressing CT-GFP; (B,D,F) CLSM images of the same suspension cells. (B) Projection of 83 images collected at 0.5 µm intervals. (D) Projection of 25 images imaged at 1 µm intervals. (F) Single image. (G) ‘Millipede- or octopus-like’ plastid structure in two tobacco suspension cells; one cell shows a side view of the nucleus, the other cell is a view from above; projection of 64 images taken at 0.5 µm intervals. Stromules are indicated by arrowheads and plastid bodies by arrows. Bars, 10 µm. A movie of a complete z-stack and of a rotating 3-D reconstruction of a cell with ‘millipede- or octopus-like’ plastids can be viewed at http://www.mbg.cornell.edu/kohler/ kohler_JCS.html. Plastid and stromule morphology 87

Fig. 5. GFP photobleaching to assess degree of autonomy of plastids clustered around the nucleus. CLSM images of plastids in a tobacco suspension cell expressing CT- GFP. (A and B) Projections of 36 images taken at 1 µm intervals along the optical z-axis. (A) Pre-bleach image; (B) Post-bleach image collected after the time-lapse; (C) Single images of a time-lapse series collected after photobleaching of the area indicated by a box in A. The area was photobleached with 100 scans at full laser power. The first image was taken 80 seconds after the beginning of photobleaching. The time-lapse images in C were taken at 20 second intervals over a period of 12 minutes. Shown are selected images with the time after the first image indicated in seconds.

Our observations of remarkable variations in shape and sizes of plastids in non-green transgenic plant tissues and cell cultures are consistent with reports of pleomorphic plastids in wild-type non- green cells, observed both by light and electron microscopy. By phase-contrast microscopy, Suzuki et al. (1992) were able to observe ‘elongated and string-like’ plastids similar to those shown in Fig. 4. Nishibayashi and Kuoriwa (1982) found that living onion epidermal cells contain leucoplasts with irregular and dynamic shapes, describing them as ‘oval, dumbbell, beads, rod frypan.’ Steffen (1964) reported the presence of filamentous and ‘ameboid’ chromoplasts in lily petals, and Whatley and Whatley (1987) described pleomorphic chromoplasts in petals rather uniform regular morphology throughout petal of a number of species. In elegant studies of plastid development (Pyke and Page, 1998). It is evident that GFP development in bean roots, consistent with our observations in labelling of plastids will allow further studies of developmental tobacco roots, Whatley (1983) documented a progression of changes in shape, number, and position in non-green cells that shape changes from spherical to ameboid non-green plastids to have previously been possible only in certain amenable cell the classic disc morphology of the mature chloroplast. Three- types in particular species. dimensional reconstruction of electron microscopic sections of We do not know how much communication occurs between pine secretory cell leucoplasts by Charon et al. (1987) showed plastids within the same cell. Our studies have conclusively they had ‘complex shape with many arms.’ In contrast to these reports of pleiomorphic plastids, leucoplasts and chloroplasts in petals of Arabidopsis were found to exhibit a

Fig. 6. Individual CLSM images of GFP-labeled plastids clustered around a nucleus in a suspension cell compared to the composite projection. (A) Projection of 31 images taken along the optical z-axis at 0.5 µm intervals; (B,C,D) Single images number 8, 9, and 10 of the stack in A. Small arrows indicate plastids that seem not to be connected to other plastids in the particular plane of focus, the large arrow points to a non-fluorescent in plastids, and the arrowhead indicates an area enclosed by a branched stromule. Bar, 10 µm. 88 R. H. Köhler and M. R. Hanson

plastid introduced into the same cell by protoplast fusion. Most somatic plants contain one or the other plastid , evidently as a result of segregation of different plastid types during regeneration (Hanson et al., 1985). By imposing selection for drug resistances carried by each of the parental plastid genomes, Medgyesy et al. (1985) were able to obtain somatic hybrids carrying both resistance , resulting from recombination of plastid genomes originally separate in different organelles. However, the frequency of recombinant plastids was extremely low. This suggests that the entire plastid genome is rarely, if at all, transmitted through stromules. Plastid DNA in vascular plants has been reported to be membrane- bound, but organization and attachment is still not well characterized (reviewed by Gillham, 1994). Perhaps chloroplast DNA, organized in , is restricted from movement Fig. 7. GFP photobleaching to assess flow of GFP through individual through the narrow stromules that sometimes connect different stromules. A selected area indicated by a white box in image A was vascular plant plastids. In contrast, Osafune et al. (1993) bleached by scanning 30 times at full laser power. A portion of reported immunolocalization experiments indicating that stromule (a) exhibits loss of fluorescence though outside the zone of chloroplast DNA is present in the cell-cycle-specific bridges bleaching, indicating flow of GFP between the bleached and that form between Euglena gracilis chloroplasts. unbleached region. Stromule (b) does not lose fluorescence in any While the vascular plant plastid genome may normally be part. Stromule (c) does not lose fluorescence in the outer part, indicating it is not connected to a stromule in the bleached zone. Bar, immobilized, unable to venture through stromules, it is possible 10 µm. (A) Pre-bleach image; (B) Cell after bleaching. that smaller DNA molecules might be able to be transmitted from one to another plastid. Following bombardment with containing an antibiotic resistance gene controlled by shown that one plastid can be connected by a tubular extension plastid gene regulatory sequences, tissue can be grown on to one or a few other plastid bodies within the same cell at a selective medium, and cells can be obtained that contain many single moment in time (e.g. Fig. 3C and Fig. 6A). We have plastids carrying the resistance gene, though presumably only shown that GFP can flow through a stromule that connects two one chloroplast originally incorporated the bombarded root plastids (Köhler et al., 1997) and that GFP can flow through DNA (Svab and Maliga, 1993). In addition to multiplication of stromules of suspension cells (Fig. 7) but we do not know about the resistant chloroplast perhaps the transforming plasmids, flow of other , small molecules, or RNA. We do not much smaller than the chloroplast genome, can move through know how often these connections break and reconnect to other stromules to plastids within the same cell. Movement of plastid bodies. In projections of CLSM images of suspension plasmids could explain in part the recent observations of cells, plastids appear to form one big interconnected unit Knoblauch et al. (1999), who were able to inject a single because of the clustering of plastid bodies around the nucleus chloroplast with a GFP gene controlled by plastid gene and occasional branching of plastid stromules. However, a flow regulatory sequences. Green fluorescence first appeared in the of GFP from plastids localized in one half of the cell into the injected chloroplast, but then became evident in other plastids other half could not be observed after photobleaching indicating within the same cell. Perhaps plasmid, RNA or protein Ð or that the majority of plastids was not interconnected during the possibly all three types of molecules Ð was transmitted from time of the experiments. Until longer time-lapse studies are one chloroplast to another. performed, we will not know whether there is a point during The role of stromules in the functioning of the plastid remains development or the cell cycle when all plastids of a particular unknown. In Acetabularia, a role of the long plastid tubules in cell type are connected simultaneously. In Euglena, connections chloroplast motility has been proposed (Menzel, 1994). between plastids were cell cycle stage-specific (Ehara et al., Alternatively, extensions of the envelope membrane could serve 1990). In Acetabularia, tubular connections are found on to facilitate transport of substances in and out of the plastid by virtually all chloroplasts in non-synchronized cultures, and only increasing surface area. The extensive network of plastids and very few chloroplasts are not connected (Menzel, 1994). stromules in cultured cells could allow plastid-synthesized Though plastid tubules in tobacco cell culture cells often molecules to be delivered to the entire cell or could assist uptake appeared to be continuous, some of our photobleaching of cytoplasmic molecules by the plastid from diverse regions of experiments revealed that GFP does not always flow from an the cell. GFP-labeled stromules frequently surround non- unbleached tubule into a bleached tubule that appeared to be fluorescent areas or particles that might be mitochondria or connected, indicating that the plastid tubules were independent (data not shown; Köhler et al., 1997). Amoeboid rather than interconnected. Therefore, caution must be used plastids in Phaseolus vulgaris root tips frequently encircle in inferring interconnectedness from images of fluorescent with ER, and mitochondria (Newcomb, plastids and stromules without confirmation by photobleaching. 1967). Perhaps close association of stromules with other Conversely, sometimes plastids apparently not connected may organelles or subcellular structures enhances the transfer of actually be bridged by a stromule that lacks sufficient GFP to molecules from one to another. If so, the frequent be detectable. occurrence of plastids and stromules in close proximity to The genetic independence of plastids has previously been nuclei could be an indication of one or two-way exchange shown by the low frequency of recombination between different between them. 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