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Exchange of Protein Molecules Through Connections Between Higher Plant Plastids

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Science  27 Jun 1997:
Vol. 276, Issue 5321, pp. 2039-2042
DOI: 10.1126/science.276.5321.2039

Abstract

Individual plastids of vascular plants have generally been considered to be discrete autonomous entities that do not directly communicate with each other. However, in transgenic plants in which the plastid stroma was labeled with green fluorescent protein (GFP), thin tubular projections emanated from individual plastids and sometimes connected to other plastids. Flow of GFP between interconnected plastids could be observed when a single plastid or an interconnecting plastid tubule was photobleached and the loss of green fluorescence by both plastids was seen. These tubules allow the exchange of molecules within an interplastid communication system, which may facilitate the coordination of plastid activities.

Plastids are plant cell organelles that perform metabolic and biosynthetic reactions, including carbon fixation and synthesis of fatty acids, carotenes, purines, and pyrimidines. Plastids contain multiple copies of a genome that encodes a subset of the organelle's RNA and protein molecules (1). Nuclear-encoded proteins are synthesized in the cytosol as precursors with transit peptides that target them to and across the chloroplast double-membrane envelope (2). Plastids are generally regarded as autonomous organelles that multiply by division and sort into daughter cells (1). We have made some observations that indicate that vascular plant plastids are less independent than previously thought.

We targeted a modified GFP (3, 4) to plastids of tobacco and petunia plants (5) by expression of a chimeric nuclear gene (4) in which the GFP coding region was connected to the transit peptide from the Arabidopsischloroplast recA gene (Fig.1A). The recA transit sequence resulted in the targeting of GFP to the stroma, the aqueous matrix of the plastid. When leaf cells were disrupted and chloroplasts were separated on a Percoll gradient (6) and examined by confocal laser scanning microscopy (CLSM) (7), intact chloroplasts exhibited the green fluorescence characteristic of GFP and the red autofluorescence of chlorophyll (Fig. 1B). Purified chloroplasts were disrupted by hypo-osmotic shock and separated into membrane and soluble portions (8). The broken chloroplast membrane fraction, which contains envelope and thylakoid membranes as well as intact thylakoids, still exhibited red (chlorophyll) fluorescence but had lost all GFP fluorescence (Fig. 1B). In immunoblots (Fig. 1C), the GFP signal was located primarily in the soluble fraction, as was anthranilate synthase (AS), a stromal enzyme (9). The membrane fraction contained the light-harvesting chlorophyll a/b protein (LHCP) (10) and a very weak GFP signal that probably resulted from contamination of the membrane fraction with stromal proteins. In cells of transgenic plants, GFP fluorescence was only visible within chloroplasts [(11) and Fig. 2].

Figure 1

(A) Chimeric gene construct (4) used to target GFP to plastids. 35S35S, double 35S promoter; CT, recA transit peptide: S65TmGFP4, GFP modified coding region; NOS, nopaline synthetase terminator region. After being processed by the transit peptidase, 15 amino acids derived from maturerecA (regular type) and the adaptor sequence (bold type) are predicted to constitute the NH2-terminal portion of the transgenic GFP. (B) Purified chloroplasts from a transgenic plant, imaged by CLSM (7). (a) Green channel, (b) merged image, and (c) red channel. (C) Chloroplast fractionation to determine the location of GFP. An immunoblot of protein fractions of an untransformed control plant (control) and of a transgenic plant (CT-GFP), probed with antibodies reacting with three different proteins, is shown. Lane 1 shows 50 μg of protein from a crude chloroplast fraction. Lane 2 shows 20 μg of protein from purified chloroplasts. Lane 3 shows 20 μg of soluble proteins from purified chloroplasts. Lane 4 shows 20 μg of insoluble membrane fraction of purified chloroplasts.

Figure 2

Tubules and tubular interconnections between chloroplasts of transgenic plants, imaged by CLSM. (A) Petunia epidermal cell. Projection of 24 optical sections taken at 0.1-μm intervals along the optical axis. (B) Tobacco trichome cell. Projection of 15 optical sections taken at 0.2-μm intervals. (C) Tobacco mesophyll cell. Projection of 36 optical sections taken 0.2 μm apart. Scale bars, 10 μm.

Thin green tubules emanating from chloroplasts are visible in leaf tissue in the transgenic plants when the tissue is examined with a standard epifluorescence microscope or by CLSM [(11) and Fig. 2]. Tubules are visible in all tissues studied so far but vary in abundance within a given cell type and between different tissue types. In some cells, tubules are evident on most chloroplasts, but in the majority of leaf cells, only a few or no tubules are visible. The tubular projections arise initially as protuberances from the chloroplast surface that elongate and extend (Fig.3). They are dynamic in the living cell, continuously changing their shape, moving around, shrinking back, and sometimes connecting with other chloroplasts. The tubules do not contain chlorophyll that is detectable by autofluorescence. Their width appears to range from 0.35 to 0.85 μm, and they may be as long as 15 μm [Fig. 2 and (11)]. Sometimes a plastid protuberance extends and appears to encircle a nonfluorescent body; this is similar to a finger curled around a sphere.

Figure 3

Tubule in a wild-type plant cell (18). (A through D) Tubule extending from the chloroplast surface in a living spinach mesophyll cell. Images were taken at about 30-s intervals. Scale bar, 10 μm.

To determine whether exchange of protein occurs through plastid interconnections, we used a two-photon laser scanning microscope (12) to selectively photobleach individual plastids or individual interconnecting tubules in living plant cells (Fig.4). When the laser was targeted to an individual plastid, loss of the GFP signal was restricted to this individual plastid, and the plastid remained photobleached after irradiation ceased during the following 35 s of observation (Fig. 4A). The synthesis and import of GFP from the cytoplasm are not sufficiently rapid to restore fluorescence over this time period. After the irradiation, nearby plastids that were not connected to the irradiated plastid remained brightly fluorescent (Fig. 4A). When the laser beam was targeted to an interconnecting chloroplast tubule (Fig. 4B), both interconnected plastids exhibited some photobleaching after 2.5 s of irradiation. A further 2.5 s of irradiation resulted in further loss of GFP fluorescence in both plastids (Fig. 4B). This indicates a rapid flow of GFP through the tubular interconnection between the plastids. We also revealed the flow of GFP from one plastid to another by photobleaching one of two interconnected plastids (Fig. 4, C and D). A 2-s irradiation reduced the GFP fluorescence in the irradiated plastid to ∼30% of the initial value. During the next 7 s, GFP fluorescence in the irradiated plastid partially recovered to 53% of the initial value; simultaneously, GFP fluorescence in the connected plastid decreased to ∼60% of its initial value, which indicates flow and equilibration of unbleached GFP between the nonirradiated plastid and the bleached plastid.

Figure 4

Localized two-photon photobleaching (12) of GFP in tobacco root plastids. Jagged arrows indicate regions that will undergo localized photobleaching. Straight arrows indicate plastids after photobleaching. Scale bars, 5 μm. (A) Control photobleaching of an unconnected plastid. (Left) The plastid at the jagged arrow in the left panel was photobleached for 2 s with an 800-nm pulsed excitation (100 fs pulse width). (Middle) The plastid immediately after bleaching (∼0.5 s). (Right) The plastid 35 s after bleaching. (B) Depletion of GFP by localized photobleaching of a connecting tubule. (Left) A 5-μm line (800 nm, 100 fs) was scanned across the interconnection at the jagged arrow for 2.5 s. (Middle) The image acquired after the first photobleaching shows the depletion of GFP in both connected plastids. (Right) Image acquired after an additional 2.5-s line scan of connecting tubule, which shows a further depletion of GFP fluorescence. (C) Photobleaching of GFP fluorescence of one plastid of a connected plastid pair. (Left) Prebleach image; a 5-μm line was scanned across the plastid at jagged arrow for 2.5 s. (Middle) The image 0.6 s after bleaching. (Right) The image 6 s after bleaching; the signal in the bleached plastid has partially recovered, whereas the signal in the connected plastid has decreased. (D) Fluorescence recovery curve from the image time series shown in (C). Four images were acquired at 0.210-s intervals, separated by a 2.2-s pause, for a total of 12 images. The plot shows the mean value from a 30 by 60 pixel box around the bleached plastid (❏), the connected unbleached plastid (❈) normalized to the prebleach image values [left panel in (C)], and the concurrent changes in the signal in the coupled plastids relative to their initial value.

Tubules emanating from the chloroplast are also observable in nontransgenic wild-type spinach and tobacco [Fig. 3 and (11)] and have been described in a variety of other wild-type vascular plant species (13, 14). Light microscopic studies and a movie made by Wildman and his colleagues (13) show dynamic protuberances extending from leaf chloroplasts of tobacco and spinach that sometimes connect to other chloroplasts. Protuberances emanating from chloroplasts have also been imaged in different species by electron microscopy (14). Because interpretation of these structures was difficult, their existence has not been widely known or accepted.

Interconnections between chloroplasts have also been detected inEuglena and Acetabularia (15, 16), although chloroplasts in these unicellular organisms differ from those of vascular plants (1). In Acetabularia, chloroplast tubules that sometimes extend over hundreds of micrometers between chloroplasts are common (15); except for their length, these tubules appear quite similar to the tubules we describe in higher plants. In Euglena, tubules connecting chloroplasts appear at specific times of the cell cycle, shortly before chloroplast division (16). Cell cycle stage–specificity of chloroplast tubules in tobacco and petunia was not investigated in our experiments, but obvious differences exist in the abundance of tubular projections in individual cells of the same tissue.

Chloroplasts are thought to have arisen from cyanobacterial endosymbionts (1, 17). The tubular connections that we observed are reminiscent of bacterial pili. Further analysis may reveal whether the tubules have any functional or compositional similarity to structures that allow the exchange of macromolecules between bacteria.

A number of functions can be envisioned for the stroma-containing interconnecting tubules. They may allow plastids to share molecules such as metabolic intermediates, nucleic acids, enzymes, and regulatory proteins. In Acetabularia, it has been proposed that the tubules support chloroplast motility (17). In vascular plants, the tubules may permit the plastid to sample volumes of the cell in which concentrations of useful molecules are higher than in the region next to the plastid body, especially if the tubules encircle other organelles such as mitochondria.

  • * To whom correspondence should be addressed. E-mail: mrh5{at}cornell.edu

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