Fluorescent Protein Aided Insights on Plastids and their Extensions: A Critical Appraisal

Abstract

Multi-colored fluorescent proteins targeted to plastids have provided new insights on the dynamic behavior of these organelles and their interactions with other cytoplasmic components and compartments. Sub-plastidic components such as thylakoids, stroma, the inner and outer membranes of the plastid envelope, nucleoids, plastoglobuli, and starch grains have been efficiently highlighted in living plant cells. In addition, stroma filled membrane extensions called stromules have drawn attention to the dynamic nature of the plastid and its interactions with the rest of the cell. Use of dual and triple fluorescent protein combinations has begun to reveal plastid interactions with mitochondria, the nucleus, the endoplasmic reticulum and F-actin and suggests integral roles of plastids in retrograde signaling, cell to cell communication as well as plant-pathogen interactions. While the rapid advances and insights achieved through fluorescent protein based research on plastids are commendable it is necessary to endorse meaningful observations but subject others to closer scrutiny. Here, in order to develop a better and more comprehensive understanding of plastids and their extensions we provide a critical appraisal of recent information that has been acquired using targeted fluorescent protein probes.

Keywords: fluorescent proteins; photoconvertible fluorescent protein; plastids; retrograde signaling; stroma; stromules.

Figure 1

Representative images of fluorescently highlighted plastids and some sub-plastidic features. (A) A top-down view of epidermal and mesophyll chloroplasts in the upper epidermis of a green house grown Arabidopsis plant expressing the stroma-targeted tpFNR:GFP. Panel “a” shows the green fluorescent stroma (488 nm excitation; emission collected—509–520 nm). Panel “b” shows chlorophyll fluorescence in red (emission band 650–750 nm) in guard cells (gc), pavement cells (pc; arrowheads in b,d), and mesophyll cell (m) chloroplasts. Note the difference in size and the GFP signal intensity between the epidermal and mesophyll chloroplasts. (B) A view of thin stroma-filled tubules (stromules; st) and the bulky, grana-containing plastid body (pb) in epidermal chloroplasts of tobacco. (C) Starch grains (sg) in mesophyll chloroplasts highlighted in an Arabidopsis plant expressing a granule bound starch synthase (GBSS) fused to GFP. (D) Clusters of plastoglobuli (pg) observed in senescent leaves of Arabidopsis expressing a Fibrillin4:mEosFP fusion. (E) The highlighting of nucleoids in chloroplasts is indicated in a transgenic Arabidopsis plant expressing a plastid envelope DNA-binding (PEND) GFP fusion. (F) View of gerontoplasts in senescent leaves in an Arabidopsis plant expressing stroma-targeted tpFNR:GFP shows their swollen appearance suggesting compromised envelope membranes, degrading chlorophyll, the presence of starch grains (sg)visible as dark non-fluorescent regions and clusters of senescence associated vesicles (sav) containing fluorescently GFP-labeled storm content. Chlorophyll auto-fluorescence in (B–F) is false colored blue. Size bars = 5 μm in (B,C); 10 μm in (A,D,E,F).

Figure 2

Analysis of time-lapse image series of chloroplasts suggests that the terms chloroplast protrusions (CP) and stromules merely represent varying degrees of plastid extension. (A) A snapshot pointing to three chloroplasts (chlorophyll depicted in blue; stroma-targeted GFP-green) in a single cell where plastid 1-does not exhibit any extension; based on a shape index (Holzinger et al., 2007b) plastid 2-exhibits small protrusions that are labeled CP; plastid 3-exhibits a clear tubular stromule (s). (B) Ten sequential images and their skeletonized version to show the plastid boundary have been taken from a time-lapse series of a single chloroplast from a plant expressing tpFNR:GFP (Movie 1). Depending upon which frame is being looked at the different stroma-filled (false colored orange) extensions and the plastid profile might be interpreted either as showing a CP (e.g., panels 1, 2, 3, 7, 8 marked with ^*) or a stromule (panels 4, 5, 6, 10 marked with S). Panel 9 (^**) shows two projections, the longer one suggesting a stromule while the shorter suggests a CP. Chlorophyll auto-fluorescence is false colored green. Size bar = 5 μm. (C) Graphic depiction of the continuously changing shape index of a single extension from a chloroplast. The extension was measured in each frame of a time-lapse video (Movie 1) as the ratio of the stromule length to it's radius at the base. Using static snapshots Holzinger et al. (2007b) had demonstrated that the average shape indexes may be grouped into two populations, one that averaged 0.8 ± 0.3 and the other at 7 ± 1.3. As analyzed here for a time-lapse series, over time a single extension can grown and shrink to span both of these categories.

Figure 3

The use of a stroma targeted green to red photo-convertible mEosFP for differential coloring of plastids allowed the long-standing idea of plastid-interconnectivity through stromules to be reassessed. (A) A row of single cells showing leucoplasts in a tobacco BY2 cell line expressing the tpFNR:mEosFP shows the three colors (green, red, yellow) that are typically achieved using the probe. Non-photoconverted plastids and stromules appear green; after a 5–7 s exposure to 490 ± 30 nm light fully photoconverted leucoplasts appear red while yellow plastids are obtained after a short 2–5 s photoconversion period. (B) Chloroplasts in a pavement cell of a stably transformed Arabidopsis line expresing stroma-targeted tpFNR:mEosFP and chlorophyll (false colored blue) with extended stromules that appear to be interacting. Prolonged observations of hundreds of similar, differentially colored, dynamic plastids and stromules failed to show protein exchange between the chloroplasts. (C) Two perspectives of the plastid are presented. Perspective A interprets it as a single, elongated plastid with a narrow intervening tubular region such as that observed during normal pleomorphy of dynamic etioplasts, chromoplasts, and leucoplasts. This perspective is favored by Schattat et al. (2012a,b, 2015). Perspective B underlies the assumption of “interconnected plastids” and considers the narrow intervening region to be a stromule that connects two bulged domains considered as two independent plastid bodies. Leucoplasts with a very similar morphology were used in FRAP experiments to establish the idea of FP flow between plastids (Köhler et al., 1997). Whereas independent plastids actually becoming interconnected have not been observed the flow of a fluorescent protein from one point to another within a single, continuous, membrane bound compartment as depicted here can hardly be disputed. Size bar: A = 25 μm; B = 5 μm.

Figure 4

Visualization of different colored FP to specific organelles facilitates investigations on plastid interactions. (A) Confocal image of chloroplasts (chlorophyll autofluorescence false colored blue) and RFP-highlighted ER shows the ER-cage around plastids in a stable transgenic Arabidopsis line. (B) An Arabidopsis line co-expressing stroma-targeted tpFNR:GFP [green; plastid body (pb) with chlorophyll false colored blue] and RFP targeted to the ER allowed the stromule (s) -ER correlation to be investigated (Schattat et al., 2011a,b). (C) A stable transgenic line coexpressing stroma targeted tpFNR:YFP and mito:GFP (Logan and Leaver, 2000) is allowing an investigation on the mitochondria (m) relationship to chloroplasts (ch) and stromules (s). (D) Investigations on F-actin (mf) relationship to chloroplasts (ch) and stromules (s) are being facilitated through a double transgenic line expressing GFP:mTalin (Kost et al., ; green) and tpFNR:mEosFP (red). F-actin around the nucleus (n) is apparent. (E) A small region from a hypocotyl cell of a triple transgenic expressing RFP targeted to the ER (er), GFP targeted to mitochondria (m) and a YFP targeted to peroxisomes (p). Chloroplasts (ch) are discernable due to their autofluorescence. The line is being used for investigating the relationship between the four organelles. (F) A double transgenic line co-expressing tpFNR:GFP and RFP-ER shows the peri-nuclear ER cage and the cluster of chloroplasts (ch) surrounding the nucleus (n) in a hypocotyl cell from a dark grown seedling. The probes might provide several interesting observations and insights into retrograde signaling between plastids and the nucleus. Size bars: A–C,E,F = 5 μm; D = 10 μm.

Figure 5

Some of the artifacts resulting from overexpression of a fusion protein. (A) Overexpression of a N-CHUP:GFP fusion results in sticky plastid envelopes and their massive clumping. (B) N-CHUP:GFP overexpression may also result in ectopic protrusions resembling stromules. Whether all such protrusions are actually stromules remains to be determined. (C) OE of FIB4:mEosFP that normally localizes to plastoglobuli (Figure 1D) can also produce localized artifacts such as extra lining of the inner membrane of the envelope. Observation made using transient expression in tobacco cells. (D) Leakage of stroma-targeted FP due to pressure/touch—induced damage to the cell makes the cytoplasm fluoresce due to mis-localization. Note the presence of chloroplasts in pavement cells. Size Bar = 5 μm in (A,B); 10 μm in (C); 50 μm in (D).