Correlated behavior implicates stromules in increasing the interactive surface between plastids and ER tubules

Stromules are extended by plastids but the underlying basis for their extension and retraction had not been understood until recently. Our live-imaging aided observations on coincident plastid stromule branching and ER tubule dynamics open out new areas of investigation relating to these rapid subcellular interactions. This addendum provides a testable hypothesis on the formation of stromules, which argues against the need for new membrane incorporation and suggests that stromal extensions might result from a remodeling of the plastid envelope membrane in an ER aided manner.Key words: stromules, plastids, endoplasmic reticulum, fluorescent proteins, subcellular interactions, FNR-EGFP, RFP-ERThe extension and retraction of stromules (stroma-filled tubules) from both chlorophyll containing and achlorophyllous plastids is well established for diverse plant species.¹ Many different conditions such as increased subcellular redox stress,² symbiotic interactions,^3–5 elevated temperatures,⁶ viral infection⁷ and alterations in plastid size and density^8,9 have been associated with stromule formation. Stromules extended from different plastids have been observed as forming connecting bridges^10–13 through which an exchange of proteins has been demonstrated.^{10,11,14–16} The latter observations strongly suggested stromules as transient communication channels between plastids for exchanging metabolites.^10,11 The connectivity of plastids via stromules also led to the suggestion that plant cells might contain a plastidom, defined as a well-connected plastid-compartment.¹² However, as pointed out by Netasan and co-workers¹⁷ most stromules do not appear to interconnect plastids and thus the movement of macromolecules between plastids might not be their sole function. In concurrence with this viewpoint the observations of Gunning¹⁸ and Lütz and Engel¹⁹ suggest that stromules have a role in increasing plastid interactions with mitochondria and peroxisomes. Thus a more generalized and acceptable statement is that stromules serve to increase the plastid stromal surface area that is exposed to the subcellular environment.Notably, the largest membrane surface area within a cell is provided by the endoplasmic reticulum (ER). Transmission electron micrographs often suggest a close proximity between plastids and the ER^20,21 and the presence of a chloroplast envelope associated-ER has been demonstrated.²² However, studies aimed at uncovering possible dynamic interactions between stromules and the ER in living plant cells had not been carried out. Our recent work²³ investigated this possible relationship by simultaneously visualizing stromules (labeled using a ferredoxin NADP(H) oxidoreductase (FNR) transit peptide fused to enhanced GFP; hereafter referred to as FNR-EGFP) and the ER (highlighted using a chimeric red fluorescent protein (RFP) carrying a basic chitinase signal sequence and an ER retention sequence; referred to as RFP-ER). Our observations clearly identified the ER as providing the nearest set of membranes with which the plastid envelope can interact. The observations are most pertinent for the narrow cortical sleeve where the relatively large sized plastids (including chloroplasts) are pressed against the cell boundary (plasma membrane and comparatively rigid cell wall) on one side and a turgid vacuole on the inner side. This space is also traversed by the cortical ER mesh created by constantly reorganizing ER-tubules. As shown in Figure 1A the side of a plastid pressed against the plasma membrane is surrounded by a loose mesh of ER tubules while the side pressed close to the vacuolar membrane (Fig. 1B) is free of the ER cradle. The cortically located plastids display strong behavioral correlations between their stromules and the neighboring ER tubules (Fig. 1C–E). Notably stromules form triangular junctions that appear very similar to the 3-way junctions displayed by the cortical ER. The three-way junctions of the ER are cisternal locations from which tubules are extended to create ER polygons. Stromule branches are extended from the stromal triangles in a very similar manner along comparable angles. Interestingly the extension and retraction of stromules and their branches occurs in tandem with contiguous ER tubules. These observations on the coincident behavior of the two organelles indicate that stromule branching and dynamic behavior might rely upon the creation of multiple membrane contact sites (MCSs) with the ER. The MCSs could aid in stretching stromules along ER tubules while loss of contact between the two membranes could cause rapid stromule retraction. While the possibility of identifying MCSs on the stromule envelope is being explored further by us our initial observations evoke a long-standing but very relevant question. Where does the membrane for forming stromules come from?Open in a separate windowFigure 1Confocal laser scanning microscopy based imaging of living Nicotiana benthamiana cells co-expressing FNR-EGFP (labels stromules) and RFP-ER (labels ER), 3D isosurface rendering and a model based on the observations. (A) 3D iso-surface rendering of a chloroplast facing the plasma membrane side shows the plastid situated in a loose cradle of ER tubules. (B) The 3D rendered chloroplast ER reconstruction seen in (A) observed from the vacuolar side after rotation by 180° shows the chloroplast appears smooth and free of the ER mesh. (C–E) Sequential frames from a time-lapse movie depict a FNR-EGFP labeled stromule undergoing a branching event. Note that in (C) the stromule extending along an ER channel with possible contacts at several points along the stromule length. (D) depicts a stromal triangle (arrowhead) that forms a branch initial. (E) depicts a branched stromule with the branches aligned with the ER (size bar = 5 µm). (F) A plastid exhibiting a short, wide stroma-filled area after stromule retraction has occured. (G) The same plastid shown in (F) without the FNR-EGFP labeled stroma (arrowhead) provides an appreciation of the “mobile jacket” created by the stromal contents around the chlorophyll containing thylakoids (size bar = 5 µm). (H and I) A diagrammatic depiction based on our observations and pertinent literature. (H) The narrow cortical region between the vacuolar membrane and the cell boundary is shared by large plastids and a dynamic ER. A depiction of the loose stroma filled jacket (arrowhead) of a plastid along with contiguous ER. Note the relative positions of the vacuolar membrane and the plasma membrane. The plastid size cannot increase isotropically within the narrow confines of the cortex and thus stromule formation along ER tubules is favored (Arrow pointing direction of stromule extension along the ER). (I) Multiple contact sites might be created between the extending stromule and the neighboring ER tubules. The diagrammatic depiction emphasizes the remodeling of the loose plastid jacket for stromule extension along ER. The schematic does not depict the strong possibility that both organelles might share an F-actin based mechanism for their extension.Stromule extension visibly enlarges the area occupied by the stromal contents of a plastid. The general conclusion of stromules increasing the plastid surface is clearly validated. Indeed Hanson and coworkers¹ calculated that for a model plastid of 3 µm diameter extending a 0.2 µm diameter and 10 µm long stromule the stromule represents about 20% of the overall plastid envelope surface area. A point that remains unclear is whether stromule extension involves a net increase in plastidic membrane or merely involves a remodeling of the existing membrane into an elongated shape. In the first scenario stromule formation would involve the creation of fresh membrane vesicles and their incorporation into an existing bi-layered plastid coat. This would be followed by their dispersal into inner and outer plastid envelopes along with complete complements of protein import and export machinery. Currently there is no experimental evidence to support this scenario. Moreover, this viewpoint does not consider observations of stromule retraction at all. If new membranes have been added during stromule extension then what happens to them during the retraction stage? Further, live imaging clearly shows that the time involved in sporadic stromule extension, branching and retraction is in the order of seconds to minutes. Such short periods do not favor the viewpoint involving creation of new membrane and its incorporation into stromules.Interestingly, observations of plastids in living cells often suggest that they have a rather wobbly form than a tight compact shape. The unstable form suggests the presence of relaxed membranes enveloping the main plastid body (Fig. 1F and G). These loose fitting membranes have been described as a “mobile jacket”^1,24 whose presence and irregular protrusions can be clearly distinguished from long stromules using a shape index.⁶ Hanson and co-workers¹ speculated that the “mobile jacket” might be stretched out and could thus provide the extra membrane needed for stromule formation. The notion is supported by the work of Gunning, which shows stromules retracting into short amorphous protrusions.¹⁸ Our observations of short protrusions or “beaks”²³ being created sporadically on a relatively regular-appearing plastid surface support these ideas. According to our observations one or two of the beaks might develop into long stromules that would be extended along neighboring ER tubules (Fig. 1H and I).Moreover, in vitro experiments on sucrose filled liposomes and giant vesicles^25,26 are particularly interesting in this context. Plasmolytic shrinkage of the sucrose filled vesicles creates states in which excess membrane surrounds a small volume. Over time the membrane exhibits small protrusions that are very similar in shape to the protrusions formed by plastid. Taken together the above-mentioned observations support behavior that might be expected upon a reshaping of already existing membranes. Notably the membrane-remodeling scenario for the formation of a stromule does not involve an increase in the net membrane of a plastid but it does achieve an increase in the surface area over which plastid stromal contents can interact with their surroundings. The viewpoint is also able to explain stromule elasticity that is suggested by their rapid extension and retraction.The new observations and ensuing discussion suggesting stromules being formed by stretching of the plastid envelope and identifying the ER as an interactive membrane partner in the process lay down the foundation for a number of other questions. These include investigations relating to the role of stromules in metabolite import and export between the two organelles, understanding the relationship between stromule and the ER behavior in relation to the underlying actin cytoskeleton as well as assessing the specificity of myosin motors that might be involved in these dynamic processes.     

Plastid stromules: video microscopy of their outgrowth, retraction, tensioning, anchoring, branching, bridging, and tip-shedding

Summary. Stromules are stroma-containing tubules which can grow from the surface of plastids, most commonly leucoplasts and chromoplasts, but also chloroplasts in some tissues. Their functions are obscure. Stills from video rate movies are presented here. They illustrate interaction of stromules with cytoskeletal strands and the anchoring of stromules to unidentified components at the cell surface. Anchoring leads to stretching and relaxation of stromules when forces arising from cytoplasmic streaming act on the attached, freely suspended plastid bodies. Data on stromule growth, retraction, and regrowth rates are provided. Formation and movement of stromular branches and bridges between plastids are described. The shedding of a tip region into the streaming cytoplasm is recorded in frame-by-frame detail, in accord with early observations. Correspondence and reprints: Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, PO Box 475, Canberra, ACT 2601, Australia.     

<Emphasis Type="Italic">In vivo</Emphasis>analysis of interactions between GFP-labeled microfilaments and plastid stromules

Background  

Plastid stromules are stroma-filled tubules that extend from the surface of plastids in higher plants and allow the exchange of protein molecules between plastids. These structures are highly dynamic; stromules change both their shape and position in the cytoplasm very rapidly. Previous studies with microfilament inhibitors indicated that stromule shape and movement are dependent on the actin cytoskeleton. To learn more about the nature of the interactions of stromules and the cytoskeleton, we imaged fluorescently-labeled microfilaments and plastids.     

New insights on stromules: Stroma filled tubules extended by independent plastids

The recognition of stromules as sporadically extended stroma filled tubules from all kinds of plastids constitutes one of the major insights that resulted from the direct application of green fluorescent protein aided imaging of living plant cells. Observations of dynamic green fluorescent stromules strongly suggested that plastids frequently interact with each other while photo-bleaching of interconnected plastids indicated that proteins can move within the stroma filled tubules. These observations readily fit into the prevailing concept of the endosymbiogenic origins of plastids and provided stromules the status of conduits for inter-plastid communication and macromolecule transfer. However, experimental evidence obtained recently through the use of photoconvertible protein labeled stromules strongly supports plastid independence rather than their interconnectivity. Additional information on stress conditions inducing stromules and observations on their alignment with other organelles suggests that the major role of stromules is to increase the interactive surface of a plastid with the rest of the cytoplasm.     

Stromule formation is dependent upon plastid size, plastid differentiation status and the density of plastids within the cell

Stromules are motile extensions of the plastid envelope membrane, whose roles are not fully understood. They are present on all plastid types but are more common and extensive on non-green plastids that are sparsely distributed within the cell. During tomato fruit ripening, chloroplasts in the mesocarp tissue differentiate into chromoplasts and undergo major shifts in morphology. In order to understand what factors regulate stromule formation, we analysed stromule biogenesis in tobacco hypocotyls and in two distinct plastid populations in tomato mesocarp. We show that increases in stromule length and frequency are correlated with chromoplast differentiation, but only in one plastid population where the plastids are larger and less numerous. We used tobacco hypocotyls to confirm that stromule length increases as plastids become further apart, suggesting that stromules optimize the plastid-cytoplasm contact area. Furthermore, we demonstrate that ectopic chloroplast components decrease stromule formation on tomato fruit chromoplasts, whereas preventing chloroplast development leads to increased numbers of stromules. Inhibition of fruit ripening has a dramatic impact on plastid and stromule morphology, underlining that plastid differentiation status, and not cell type, is a significant factor in determining the extent of plastid stromules. By modifying the plastid surface area, we propose that stromules enhance the specific metabolic activities of plastids.     

Visualisation of stromules in transgenic wheat expressing a plastid-targeted yellow fluorescent protein

Stromules are stroma-filled tubules that extend from the plastids in all multicellular plants examined to date. To facilitate the visualisation of stromules on different plastid types in various tissues of bread wheat (Triticum aestivum L.), a chimeric gene construct encoding enhanced yellow fluorescent protein (EYFP) targeted to plastids with the transit peptide of wheat granule-bound starch synthase I was introduced by Agrobacterium-mediated transformation. The gene construct was under the control of the rice Actin1 promoter, and EYFP fluorescence was detected in plastids in all cell types throughout the transgenic plants. Stromules were observed on all plastid types, although the stromule length and abundance varied markedly in different tissues. The longest stromules (up to 40 μm) were observed in epidermal cells of leaves, whereas only short beak-like stromules were observed on chloroplasts in mesophyll cells. Epidermal cells in leaves and roots contained the highest proportion of plastids with stromules, and stromules were also abundant on amyloplasts in the endosperm tissue of developing seeds. The general features of stromule morphology and distribution were similar to those shown previously for tobacco (Nicotiana tabacum L.) and arabidopsis (Arabidopsis thaliana (L.) Heynh.).     

Plastid stromules are induced by stress treatments acting through abscisic acid

Stromules are highly dynamic stroma-filled tubules that extend from the surface of all plastid types in all multi-cellular plants examined to date. The stromule frequency (percentage of plastids with stromules) has generally been regarded as characteristic of the cell and tissue type. However, the present study shows that various stress treatments, including drought and salt stress, are able to induce stromule formation in the epidermal cells of tobacco hypocotyls and the root hairs of wheat seedlings. Application of abscisic acid (ABA) to tobacco and wheat seedlings induced stromule formation very effectively, and application of abamine, a specific inhibitor of ABA synthesis, prevented stromule induction by mannitol. Stromule induction by ABA was dependent on cytosolic protein synthesis, but not plastid protein synthesis. Stromules were more abundant in dark-grown seedlings than in light-grown seedlings, and the stromule frequency was increased by transfer of light-grown seedlings to the dark and decreased by illumination of dark-grown seedlings. Stromule formation was sensitive to red and far-red light, but not to blue light. Stromules were induced by treatment with ACC (1-aminocyclopropane-1-carboxylic acid), the first committed ethylene precursor, and by treatment with methyl jasmonate, but disappeared upon treatment of seedlings with salicylate. These observations indicate that abiotic, and most probably biotic, stresses are able to induce the formation of stromules in tobacco and wheat seedlings.     

Microfilaments and microtubules control the morphology and movement of non-green plastids and stromules in Nicotiana tabacum

Plastid stromules are stroma-filled tubular extensions of the plastid envelope membrane. These structures, which have been observed in a number of species, allow transfer of proteins between interconnected plastids. The dramatic shape of stromules and their dynamic movement within the cell provide an opportunity to study the control of morphology and motion of plastids. Using inhibitors of actin and tubulin, we found that both microfilaments and microtubules affect the shape and motility of non-green plastids. Actin and tubulin control plastid and stromule structure by independent mechanisms, while plastid movement is promoted by microfilaments but inhibited by microtubules. The presence or absence of stromules does not affect the motility of plastids. Photobleaching experiments indicate that actin and tubulin are not necessary for the bulk of green fluorescent protein (GFP) movement between plastids via stromules.     

Stromules: a characteristic cell-specific feature of plastid morphology

Stromules (stroma-filled tubules) are highly dynamic structures extending from the surface of all plastid types examined so far, including proplastids, chloroplasts, etioplasts, leucoplasts, amyloplasts, and chromoplasts. Stromules are usually 0.35-0.85 microm in diameter and of variable length, from short beak-like projections to linear or branched structures up to 220 mum long. They are enclosed by the inner and outer plastid envelope membranes and enable the transfer of molecules as large as Rubisco (approximately 560 kDa) between interconnected plastids. Stromules occur in all cell types, but stromule morphology and the proportion of plastids with stromules vary from tissue to tissue and at different stages of plant development. In general, stromules are more abundant in tissues containing non-green plastids, and in cells containing smaller plastids. The primary function of stromules is still unresolved, although the presence of stromules markedly increases the plastid surface area, potentially increasing transport to and from the cytosol. Other functions of stromules, such as transfer of macromolecules between plastids and starch granule formation in cereal endosperm, may be restricted to particular tissues and cell types.     

The Xanthomonas effector XopL uncovers the role of microtubules in stromule extension and dynamics in Nicotiana benthamiana

Xanthomonas campestris pv. vesicatoria type III‐secreted effectors were screened for candidates influencing plant cell processes relevant to the formation and maintenance of stromules in Nicotiana benthamiana lower leaf epidermis. Transient expression of XopL, a unique type of E3 ubiquitin ligase, led to a nearly complete elimination of stromules and the relocation of plastids to the nucleus. Further characterization of XopL revealed that the E3 ligase activity is essential for the two plastid phenotypes. In contrast to the XopL wild type, a mutant XopL lacking E3 ligase activity specifically localized to microtubules. Interestingly, mutant XopL‐labeled filaments frequently aligned with stromules, suggesting an important, yet unexplored, microtubule–stromule relationship. High time‐resolution movies confirmed that microtubules provide a scaffold for stromule movement and contribute to stromule shape. Taken together, this study has defined two populations of stromules: microtubule‐dependent stromules, which were found to move slower and persist longer, and microtubule‐independent stromules, which move faster and are transient. Our results provide the basis for a new model of stromule dynamics including interactions with both actin and microtubules.     

TGD5 is required for normal morphogenesis of non-mesophyll plastids,but not mesophyll chloroplasts,in Arabidopsis

Stromules are dynamic membrane-bound tubular structures that emanate from plastids. Stromule formation is triggered in response to various stresses and during plant development, suggesting that stromules may have physiological and developmental roles in these processes. Despite the possible biological importance of stromules and their prevalence in green plants, their exact roles and formation mechanisms remain unclear. To explore these issues, we obtained Arabidopsis thaliana mutants with excess stromule formation in the leaf epidermis by microscopy-based screening. Here, we characterized one of these mutants, stromule biogenesis altered 1 (suba1). suba1 forms plastids with severely altered morphology in a variety of non-mesophyll tissues, such as leaf epidermis, hypocotyl epidermis, floral tissues, and pollen grains, but apparently normal leaf mesophyll chloroplasts. The suba1 mutation causes impaired chloroplast pigmentation and altered chloroplast ultrastructure in stomatal guard cells, as well as the aberrant accumulation of lipid droplets and their autophagic engulfment by the vacuole. The causal defective gene in suba1 is TRIGALACTOSYLDIACYLGLYCEROL5 (TGD5), which encodes a protein putatively involved in the endoplasmic reticulum (ER)-to-plastid lipid trafficking required for the ER pathway of thylakoid lipid assembly. These findings suggest that a non-mesophyll-specific mechanism maintains plastid morphology. The distinct mechanisms maintaining plastid morphology in mesophyll versus non-mesophyll plastids might be attributable, at least in part, to the differential contributions of the plastidial and ER pathways of lipid metabolism between mesophyll and non-mesophyll plastids.     

Isolation and analysis of a stromule‐overproducing Arabidopsis mutant suggest the role of PARC6 in plastid morphology maintenance in the leaf epidermis

Stromules, or stroma‐filled tubules, are thin extensions of the plastid envelope membrane that are most frequently observed in undifferentiated or non‐mesophyll cells. The formation of stromules is developmentally regulated and responsive to biotic and abiotic stress; however, the physiological roles and molecular mechanisms of the stromule formation remain enigmatic. Accordingly, we attempted to obtain Arabidopsis thaliana mutants with aberrant stromule biogenesis in the leaf epidermis. Here, we characterize one of the obtained mutants. Plastids in the leaf epidermis of this mutant were giant and pleomorphic, typically having one or more constrictions that indicated arrested plastid division, and usually possessed one or more extremely long stromules, which indicated the deregulation of stromule formation. Genetic mapping, whole‐genome resequencing‐aided exome analysis, and gene complementation identified PARC6/CDP1/ARC6H, which encodes a vascular plant‐specific, chloroplast division site‐positioning factor, as the causal gene for the stromule phenotype. Yeast two‐hybrid assay and double mutant analysis also identified a possible interaction between PARC6 and MinD1, another known chloroplast division site‐positioning factor, during the morphogenesis of leaf epidermal plastids. To the best of our knowledge, PARC6 is the only known A. thaliana chloroplast division factor whose mutations more extensively affect the morphology of plastids in non‐mesophyll tissue than in mesophyll tissue. Therefore, the present study demonstrates that PARC6 plays a pivotal role in the morphology maintenance and stromule regulation of non‐mesophyll plastids.     

The Formation of Stromules In Vitro from Chloroplasts Isolated from Nicotiana benthamiana

Stromules are stroma-containing tubules that have been observed to emanate from the main plastidic body in vivo. These structures have been shown to require cytoskeletal components for movement. Though numerous studies have shown a close association with the endoplasmic reticulum, nucleus, mitochondria, and other plastids, the mechanism of formation and their overall function remain unknown. A limiting factor in studying these structures has been the lack of a reconstituted system for in vitro stromule formation. In this study, stromule formation was induced in vitro by adding a plant extract fraction that is greater than 100 kDa to a population of isolated chloroplasts. Kinetic measurements show that stromule formation occurs within ~10 seconds after the addition of the plant extract fraction. Heat inactivation and apyrase treatment reveal that the stromule stimulating compound found in the extract fraction is a protein or protein complex 100 kDa or greater. The formation of the stromules in vitro with isolated chloroplasts and a concentrated fraction of cell extract opens an avenue for the biochemical dissection of this process that has heretofore been studied only in vivo.     

Endoplasmic reticulum dynamics, inheritance, and cytoskeletal interactions in budding yeast

The endoplasmic reticulum (ER) in Saccharomyces cerevisiae consists of a reticulum underlying the plasma membrane (cortical ER) and ER associated with the nuclear envelope (nuclear ER). We used a Sec63p-green fluorescent protein fusion protein to study motility events associated with inheritance of cortical ER and nuclear ER in living yeast cells. During M phase before nuclear migration, we observed thick, apparently rigid tubular extensions emanating from the nuclear ER that elongate, undergo sweeping motions along the cell cortex, and shorten. Two findings support a role for microtubules in this process. First, extension of tubular structures from the nuclear ER is inhibited by destabilization of microtubules. Second, astral microtubules, structures that undergo similar patterns of extension, cortical surveillance and retraction, colocalize with nuclear ER extensions. During S and G(2) phases of the cell cycle, we observed anchorage of the cortical ER at the site of bud emergence and apical bud growth. Thin tubules of the ER that extend from the anchored cortical ER display undulating, apparently random movement and move into the bud as it grows. Finally, we found that cortical ER morphology is sensitive to a filamentous actin-destabilizing drug, latrunculin-A, and to mutations in the actin-encoding ACT1 gene. Our observations support 1) different mechanisms and cytoskeletal mediators for the inheritance of nuclear and cortical ER elements and 2) a mechanism for cortical ER inheritance that is cytoskeleton dependent but relies on anchorage, not directed movement.     

Stromules: Mobile Protrusions and Interconnections Between Plastids

Abstract: Stroma-filled tubules, recently named stromules, extend from the surface of plastids in most cell types and plant species examined. Stromules are highly dynamic structures, continuously and rapidly changing shape. They have been shown to interconnect plastids and permit the exchange of green fluorescent protein (GFP) between plastids. Stromules are enclosed by the inner and outer plastid envelope membranes and are 0.4 - 0.8 μm in diameter and up to 65 μm long. Movement of stromules is dependent on the actin cytoskeleton and the ATPase activity of myosin. Stromules are more abundant in cells containing a relatively small plastid volume and provide a means of enormously increasing the plastid surface area. Many important questions on the structure, function and mobility of stromules remain unanswered.     

Differential coloring reveals that plastids do not form networks for exchanging macromolecules

Stroma-filled tubules named stromules are sporadic extensions of plastids. Earlier, photobleaching was used to demonstrate fluorescent protein diffusion between already interconnected plastids and formed the basis for suggesting that all plastids are able to form networks for exchanging macromolecules. However, a critical appraisal of literature shows that this conjecture is not supported by unequivocal experimental evidence. Here, using photoconvertible mEosFP, we created color differences between similar organelles that enabled us to distinguish clearly between organelle fusion and nonfusion events. Individual plastids, despite conveying a strong impression of interactivity and fusion, maintained well-defined boundaries and did not exchange fluorescent proteins. Moreover, the high pleomorphy of etioplasts from dark-grown seedlings, leucoplasts from roots, and assorted plastids in the accumulation and replication of chloroplasts5 (arc5), arc6, and phosphoglucomutase1 mutants of Arabidopsis thaliana suggested that a single plastid unit might be easily mistaken for interconnected plastids. Our observations provide succinct evidence to refute the long-standing dogma of interplastid connectivity. The ability to create and maintain a large number of unique biochemical factories in the form of singular plastids might be a key feature underlying the versatility of green plants as it provides increased internal diversity for them to combat a wide range of environmental fluctuations and stresses.     

Trafficking of Proteins through Plastid Stromules

Stromules are thin projections from plastids that are generally longer and more abundant on non-green plastids than on chloroplasts. Occasionally stromules can be observed to connect two plastid bodies with one another. However, photobleaching of GFP-labeled plastids and stromules in 2000 demonstrated that plastids do not form a network like the endoplasmic reticulum, resulting in the proposal that stromules have major functions other than transfer of material from one plastid to another. The absence of a network was confirmed in 2012 with the use of a photoconvertible fluorescent protein, but the prior observations of movement of proteins between plastids were challenged. We review published evidence and provide new experiments that demonstrate trafficking of fluorescent protein between plastids as well as movement of proteins within stromules that emanate from a single plastid and discuss the possible function of stromules.Projections from chloroplasts have been reported sporadically in the literature for over a hundred years (reviewed in Gray et al., 2001; Kwok and Hanson, 2004a) and became established as genuine features of plastids when they were observed by the targeting of green fluorescent protein (GFP) to the stromal compartment (Köhler et al., 1997). This study showed that these projections sometimes appeared to connect discrete plastid bodies, and photobleaching experiments demonstrated flow of GFP from one plastid body to another. After GFP in one plastid body was bleached, fluorescence rapidly recovered as a result of flow from GFP from the unbleached plastids. By continuous bleaching of a stromule connecting two plastids, fluorescence was lost from both plastids. This led to the speculation that there could be an interplastid communication system (Köhler et al., 1997). In a follow-up study to test the degree of interplastid connectedness, the term “stromule” was coined to prevent confusion with other tubular structures in the cell (Köhler and Hanson, 2000). The existence of a stromule-based plastid network was ruled out by these experiments, but movement of protein through stromules was confirmed, and it was proposed that stromules might function to facilitate transport of substances in and out of the plastid by increasing surface area and by placing the plastid compartment in close proximity to other organelles or subcellular structures (Köhler and Hanson, 2000). A study by Schattat et al. (2012) confirmed the absence of a plastid network with the use of a photoconvertible fluorescent protein. These authors also describe photoconversion experiments that appear to contradict our prior work demonstrating flow of GFP between two plastid bodies connected by a stromule. Here, we confirm our prior fluorescence recovery after photobleaching (FRAP) results, showing that proteins can move through stromules between individual plastids, and we demonstrate that a red photoconverted protein can also move into a region where photoconversion has not occurred, provided that potentially damaging levels of light are not used during the photoconversion experiment. We review previous studies showing the lack of an interconnected plastid network and consider other functions for stromules, such as facilitating the transport of enzymes and metabolites to and from the plastid to the vicinity of other organelles or regions of the cell.     

Dynamic morphology of plastids and stromules in angiosperm plants

Labelling of plastids with fluorescent proteins has revealed the diversity of their sizes and shapes in different tissues of vascular plants. Stromules, stroma-filled tubules comprising thin extensions of the stroma surrounded by the double envelope membrane, have been observed to emanate from all major types of plastid, though less common on chloroplasts. In some tissue types, stromules are highly dynamic, forming, shrinking, attaching, releasing and fragmenting. Stromule formation is negatively affected by treatment of tissue with cytoskeletal inhibitors. Plastids can be connected by stromules, through which green fluorescent protein (GFP) and fluorescently tagged chloroplast protein complexes have been observed to flow. Within the highly viscous stroma, proteins traffic by diffusion as well as by an active process of directional travel, whose mechanism is unknown. In addition to exchanging materials between plastids, stromules may also serve to increase the surface area of the envelope for import and export, reduce diffusion distance between plastids and other organelles for exchange of materials, and anchor the plastid onto attachment points for proper positioning with the plant cell. Future studies should reveal how these functions may affect plants in adapting to the challenges of a changing environment.     

Exclusion of plastid nucleoids and ribosomes from stromules in tobacco and Arabidopsis

Stromules are stroma-filled tubules that extend from the surface of plastids and allow the transfer of proteins as large as 550 kDa between interconnected plastids. The aim of the present study was to determine if plastid DNA or plastid ribosomes are able to enter stromules, potentially permitting the transfer of genetic information between plastids. Plastid DNA and ribosomes were marked with green fluorescent protein (GFP) fusions to LacI, the lac repressor, which binds to lacO-related sequences in plastid DNA, and to plastid ribosomal proteins Rpl1 and Rps2, respectively. Fluorescence from GFP-LacI co-localised with plastid DNA in nucleoids in all tissues of transgenic tobacco (Nicotiana tabacum L.) examined and there was no indication of its presence in stromules, not even in hypocotyl epidermal cells, which contain abundant stromules. Fluorescence from Rpl1-GFP and Rps2-GFP was also observed in a punctate pattern in chloroplasts of tobacco and Arabidopsis [Arabidopsis thaliana (L.) Heynh.], and fluorescent stromules were not detected. Rpl1-GFP was shown to assemble into ribosomes and was co-localised with plastid DNA. In contrast, in hypocotyl epidermal cells of dark-grown Arabidopsis seedlings, fluorescence from Rpl1-GFP was more evenly distributed in plastids and was observed in stromules on a total of only four plastids (<0.02% of the plastids observed). These observations indicate that plastid DNA and plastid ribosomes do not routinely move into stromules in tobacco and Arabidopsis, and suggest that transfer of genetic information by this route is likely to be a very rare event, if it occurs at all.     

Plastids and stromules interact with the nucleus and cell membrane in vascular plants

The various metabolic activities of plastids require continuous exchange of reactants and products with other organelles of the plant cell. Physical interactions between plastids and other organelles might therefore enhance the efficiency of plant metabolism. We have observed a close apposition of plastids and nuclei in various organs of Nicotiana tabacum and Arabidopsis thaliana. In hypocotyl epidermal cells, plastids and stromules, stroma-filled tubular extensions of the plastid envelope membrane, were observed to reside in grooves and infoldings of the nuclear envelope, indicating a high level of contact between the two organelle membranes. In a number of non-green tissues, including suspension-cultured cells, perinuclear plastids were frequently associated with long stromules that extended from the cell center to the cell membrane. In cotyledon petioles, cells lying adjacent to one another frequently contained stromules that met on either side of the shared cell wall, suggesting a means of intercellular communication. Our results therefore suggest that stromules have diverse roles within plant cells, perhaps serving as pathways between nuclei and more distant regions of the cell and possibly even other cells.