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

Abstract

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.

Figure 1. Green to red photoconvertible monomeric Eos fluorescent protein targeted to the plastid stroma allows differential coloring of plastids and has resulted in new insights on stromules. (A) Observing epidermal plastids highlighted through the green non-photoconverted form of tpFNR:mEosFP strongly suggests plastid inter-connectivity (1) The dotted circle in (2) indicates the dividing plastid that is illuminated for a few seconds with violet-blue light for achieving irreversible green to red photo-conversion of mEosFP. (3) Photo-conversion results in differential coloring of the plastids and facilitates investigations on stromule interactions and possible exchange of proteins. In this case (3) the red mEosFP remains exclusively in the photoconverted plastid disproving the previous assumption of plastid interconnectivity (1); (B) Etioplasts in dark grown seedlings are pleomorphic and often appear stretched whereby they can be easily misinterpreted as two or more inter-connected plastids. (1) non-photoconverted leucoplast in a root cell in A. thaliana. (2) Photoconverted etioplast in A. thaliana hypocotyl cell with three bulbous dilations along the stretched plastid (circle marking the area illuminated by violet-blue light for photoconversion); (C) Elongated or tubular plastids represent single double membrane bound compartment. Photoconversion on one end of the plastid (1), results in a rapid intermixing of the green and red forms of mEosFP to provide a uniform orange color (2). Such fluorescent protein flow within the plastid compartment is similar to that which was achieved in earlier studies^,, through the photo-bleaching of GFP in one part of a tubular plastid. Size bars = 5 µm.

Figure 2. Non-green plastids like etioplasts and leucoplasts lack large rigid internal structures and this allows their shape to be more flexible than mesophyll chloroplasts. The morphological flexibility and the formation of a constricted isthmus in late stages of plastid division frequently gives the impression of two or more plastids being connected by stromules. Because most of these plastids are missing grana organization, the identification of the main plastid body in pleomorphic tubules is difficult. Seven frames (1–7) from a 5 min time series depicting a plastid in a mature A. thaliana root epidermal cell show plastid pleomorphy. At the beginning (1), three bulbous regions are connected by tubules, suggesting plastids interconnected by stromules. However the chlorophyll signal, which can be detected in plastids in mature roots of in vitro, light grown plants, is observed in only two of the plastid shaped bulbous regions (marked by '*'). The RGB-intensity plot (see ; blue line denotes chlorophyll fluorescence) confirms the observation of two plastids only in the group of bulbous dilations. During the time lapse series the dilation without chlorophyll signal fused with one of the chlorophyll signal-containing bulbs (2–4), indicating that both bulbs were indeed parts of the same plastid. (8) The RGB-intensity plots along the dotted line in (1) illustrate the even distribution of photoconverted red and non-photoconverted green mEosFP (visible in the merged images 1–7 as yellowish color) and clearly proves the presence or absence of chlorophyll in the bulbs (overlay of blue chlorophyll signals with green and red result in white sectors ‘*’). Prominent movements of bulbs are indicated by arrows. Size bar = 5µm.

Figure 3. Stromule frequency in epidermal cells of A. thaliana plants expressing FNR-EGFP is dependent upon illumination. The presence of light favors the formation of stromules where in darkness stromule frequency declines slowly until a base level of about 5% is reached. Nevertheless, stromules can be induced by sucrose application in dark-adapted leaves. (A-C) 24-h (h) experiments showing average stromule frequency and the respective 5% confidence intervals. Probes were taken every three hours. The normal 12h/12h day light cycle phases is marked ‘*’ ; extended phases are marked ‘**’. (D) Gray-scale images of epidermal plastids at the end of a normal day showing plastids with stromules, (E) Plastids at the end of a normal night are mainly without stromules. (F) Average stromule frequency and respective 5% confidence intervals for 40 mM sucrose treatment (gray), a prominent inducer of stromules in light adapted leaves, and water treated control (white). Size bar = 5 µm.

Figure 4. The new findings using stroma targeted photoconvertible EosFP change our idea of stromules as being interplastid conduits and instead present them as plastidic extensions for more effective communication and interaction with other cellular components such as the ER, small organelles such as mitochondria and peroxisomes (depicted in blue) and the cytosol in general. Notably, the new insight presents plastids and their stromules as pleomorphic but unique organelles and not subunits of a single homogenous plastid population. The uniqueness of each plastid might eventually be responsible for the tremendously flexible and versatile nature of plant cells.