As noted earlier by Jedd and Chua (2002), Mano et al. (2002), Mathur et al. (2002) and Collings et al. (2002) the basis for erratic movement of peroxisomes in plants has remained unexplained. The seemingly individualistic patterns of peroxisomal motility include staying in one subcellular location for varying durations, bi-directional movements, cyclic revolution within a small region of the cell, U-turns, and sudden lateral or tangential forays (Mano et al., 2002; Mathur et al., 2002). Much of the straight axial movement can be attributed to direct peroxisomal dependence on myosin motors and the underlying F-actin cytoskeleton as part of the main cytoplasmic stream (Grolig and Pierson, 2000; Jedd and Chua, 2002). However, neither myosin motors nor the existence of fine F-actin tracks directly elucidate the mechanism underlying sudden stops, oscillations and other peculiarities of peroxisomal motility (Mathur et al., 2002). Interestingly the fine cortical F-actin mesh in a plant cell also provides a structural basis for cortical ER organization in plants (Quader and Zachariadis, 2006; Runions et al., 2006; Sparkes et al., 2009) and thus we speculated that observing the simultaneous behavior of peroxisomes and the ER might provide insights on the erratic motility of peroxisomes.

The cortical ER in plant cells displays a dynamic pattern of non-uniform polygons created by anastomosing cyto-membranes wherein smooth tubules and lamellar segments undergo constant rearrangement, sliding, branching and fusion. Sub-cortically, the ER forms long tubules and fenestrated sheets that enmesh different organelles and form a major component of cytoplasmic strands stretching across the large, vacuolated plant cell (Quader and Zachariadis, 2006). Observations on dual marker lines co-expressing ss-mGFP5-HDEL and YFP-PTS1 as well as the YFP-RFP dual combination revealed many instances of forward movement followed by a U-turn with constant variation in the rate of motility. A representative time-lapse series is provided (Figures 1A,B) where the movement of a single peroxisome (Figure 1C—peroxisome with red spot) is charted (Figure 1A frames 1–22) and presented as a merged image (Figure 1B). The variable distance moved between each frame is presented in Figure 1C whereas Figure 1D follows the movement carried out over 120 s for 6 independent peroxisomes. Clearly, different peroxisomes move differently and there is a range of variation in their rate of their movement (Figure 1D). As observed in the images (and the Movie S1) the motility of the peroxisome closely followed the patterns of the neighboring ER (Figure 1A). Another representative series of 13 sequential images (Movie S2) each separated by ca. 8 s (Figure 1E frames 1–13) follows the movements of 3 peroxisomes (Figure 1E; 1-a, b, c) and reveals that to-and-fro oscillations ranging between 2 and 8μm usually occur along short ER-tubules extending and retracting from an ER junction (1E; peroxisomes a, b frames 1–8). As shown in Figure 1E (frames 8–10) such movements continue until the ER tubule harboring a peroxisome fuses with another ER tubule to create the familiar ER polygon, whereupon the peroxisome moves again in the pattern defined by the newly organized polygon (Figure 1E; peroxisome b; frames 7–10). Finally the oscillating peroxisomes get drawn into a fast moving cytoplasmic ER strand and move away rapidly from their previous locations at velocities approaching 4 ± 1.5μms⁻¹ (Figure 1E frames 9–13).

The cortical ER also consists of large membrane patches that constitute ER islands. When observed using only YFP-PTS1 many peroxisomes appear moribund or execute only Brownian movement (Mathur et al., 2002). When co-visualized with the ER these peroxisomes were confined to the ER islands (e.g., peroxisome-c in Figure 1E). These observations of individual peroxisomal activity within different regions of the interlinked and constantly rearranging ER suggested an explanation for the seemingly erratic peroxisome motility. The observations also suggested how, depending upon the rate of ER motility, up to 65 ± 3% (n = 250) peroxisomes within a cell might appear to be arrested in a particular sub-cellular location while other peroxisomes move past them at varying speeds. We further investigated this line of thought by observing peroxisomal motility under conditions that are known to affect ER dynamics.

Low temperature, around 4°C slows down ER motility, leads to the disappearance of long tubular ER strands and causes a distinct increase in short ER tubules and wider cisternae (Quader et al., 1989). Seedlings expressing RFP-ER and YFP-PTS1 were cold treated for 6 h and analyzed for motility. Figure 2A shows the change in peroxisome motility over 12 min as the ambient temperature around the cold treated seedlings returned to 23°C. Hypocotyl epidermal cells visualized within 3 min of removal from ice-cold temperatures displayed short cortical ER tubules, slow tumbling movements of spindle shaped ER bodies and a low motility of the ER in cytoplasmic strands. Only 2.79 ± 0.9% peroxisomes (n = total 100 peroxisomes observed in 10 different cells in 10 seedlings) displayed short oscillations in these cells with the rest exhibiting Brownian movement. By 6 min the number of motile peroxisomes had risen to 35 ± 2.8%, while ER-tubules started exhibiting remodeling and new areas of anastomosis to form the typical polygonal ER mesh. By 12 min the ER exhibited normal cortical and sub-cortical dynamics and 67 ± 5% peroxisomes had achieved motility with normal speeds of 4 ± 1.5μms⁻¹ in sub-cortical strands. It was concluded that while slowing down the dynamic behavior of the ER results in slowing peroxisome motility an increase in the movement rate of the ER results in a concomitant change in peroxisomal movement.

Both actin and myosin inhibitors have previously been shown to interfere with peroxisome (Collings et al., 2002; Jedd and Chua, 2002; Mano et al., 2002; Mathur et al., 2002) and ER (Liebe and Quader, 1994) motility. Both organelles respond similarly by displaying an arrest of motility that is reversible upon washing out the inhibitor. The visualization of their concomitant response to 1μM Latrunculin-B is shown in Figure 2B. In all hypocotyl cells ER and peroxisome motility ceased within 5 min of being exposed to the inhibitor. Both organelles froze in place but did not lose their form. However, in some areas peroxisomes were seen aggregating around large ER blobs (Figure 2B; asterisk). After 10 min the inhibitor was washed away through 5 sequential washes with 10 ml water. Within 5 min of the washing clear indications of ER recovery including the extension-retraction of cortical ER tubules, the resumption of sub-cortical-ER flow in cytoplasmic strands and the stirring of ER bodies, were observed. Concomitantly, as shown through a representative series of time-lapse images the range of peroxisomal oscillations alongside ER tubules started increasing. In this early recovery period covering 3 min 80 ± 6% (n = 200) peroxisomes displayed relatively localized movements. These involved multiple circumambulations (path shown in Figure 2C frame 10) with diameters ranging from 10 to 15μm before single peroxisomes moved away on tracks defined by tangential ER tubules (Figure 2C frames 1–10). By 10 min after inhibitor removal a majority of cells displayed normal ER and peroxisomal motility. The drug treatments in our experiments were restricted to 10 min since longer exposures to 1μM Lat-B result in increased ER cisternae and aggregation (Figure 2D) where upon the associated population of peroxisomes becomes limited to Brownian movement. As estimated through observations carried out every 30 min more than 90% of such cells with ER globules did not recover complete organelle motility during 2 h. Similar ER-peroxisome aggregates were often observed in root cells that had been injured or been kept in water, presumably under hypoxic conditions for 2–3 h.

Our observations suggested a close correlation in the pattern of peroxisome motility and the dynamic behavior of ER tubules, but did not provide a sense of the physical relationship between the two organelles. In an earlier study (Sinclair et al., 2009) we have showed that thin tubules, called peroxules, are formed by peroxisomes exposed to subcellular oxidative stress. The peroxules are extended and retracted along ER tubules. We speculated that elongated peroxisomes would provide a better way of understanding the spatio-temporal relationship between peroxisomes and the ER in comparison to our observations on the small ca. 1μm diameter organelles. Peroxisomes in the apm1/drp3a (Mano et al., 2004) mutant were visualized for this purpose.

Peroxisomes in the apm1/drp3a mutant of Arabidopsis are unable to undergo efficient fission and break into the typically spherical, ca. 0.4–1.5μm diameter organelles. Consequently the apm1 mutants display peroxisomes with an abnormally elongated morphology (Mano et al., 2004). Time lapse imaging of GFP-highlighted elongated peroxisomes in the apm1-1 mutant showed them morphing rapidly into various contorted forms that included polygons and extensions such as those displayed by the ER (Figures 3A,B). However, in the case of the ER the polygons are part of a continuous system and undergo constant fission and fusion (Quader and Zachariadis, 2006; Sparkes et al., 2009). The resemblance of peroxisomes in the apm1-1 mutant to ER polygons and reticulum made us wonder whether the peroxisomal tubules could actually be a part of the existing ER mesh. In such a situation, we reasoned, the tubules would also be able to fuse and exchange luminal proteins. In an earlier report on plastid stromules we have used photo-convertible protein based differential coloring to demonstrate mitochondrial fusion and plastid non-fusion (Schattat et al., 2012). Mitochondrial fusion resulted in their contents mixing and resulted in an intermediate color between red and green (Schattat et al., 2012). Therefore we introduced a green to red photo-convertible EosFP-PTS1 (Sinclair et al., 2009; Figure 3C) in the T-DNA insertional mutant allele of apm1-1 (SALK_066958). The initially green fluorescent tubular peroxisomes in these lines could be readily photo-converted to red fluorescent ones through irradiation with a violet-blue light (355–425 nm; Figure 3C). We observed occasional fission of single red and green tubules but in 80 tubules from 10 different seedlings, where sometimes multiple tubules intermingled with each other for several min, we were unable to find even a single instance where a red tubular peroxisome fused with a green one. Representative frames from a time-lapse image sequence are presented (Figure 3D). We concluded that individual peroxisomes, whether spherical or tubular, maintain a closed boundary and do not re-establish luminal continuity with other peroxisomes. Having established the individuality and closed nature of tubular peroxisomes, that displayed ER like behavior, we sought to investigate their spatial relationship with ER tubules.

Whereas observations of green fluorescent tubular peroxisomes in the apm1-1 and its T-DNA insertional allele (SALK_066958) strongly suggested that one is observing an ER like compartment the simultaneous time-lapse observations of peroxisomes and the ER in double transgenics expressing GFP-PTS1 and RFP-ER showed several instances where peroxisomal tubules lay in ER lined channels (Figure 4A; Movie S3). The merged images clearly showed that the extension of tubular peroxisome closely followed the reorganization and dynamics of neighboring ER tubules (Figure 4A; frames 1–8). In other time-lapse images (such as Figure 4B frames 1–7; Movie S4) instances of a tubular peroxisome lodged on an ER island and rejoining a rapidly moving sub-cortical ER strand were noted (Figure 4B; Movie S4). These observations match the observations on motility of spherical peroxisomes in wild type plants (Figures 1A–E) but with the added advantage of being able to observe a larger area in which the two organelles maintain closeness. Occasionally the contiguity may extend to tubular peroxisomes wrapping around ER organelles such as large spindle shaped ER bodies as they pass by as part of the streaming cytoplasm (Figure 4C; Movie S5). In such cases the transient association, even if it occurs against the track being followed originally by the peroxisome (Movie S5), results in the peroxisomal shape tracing out the organelle surface (Figure 4C frames 1–7). In addition several instances were noted where a long tubular peroxisome broke into two unequal parts through the dynamic reorganization of the surrounding ER. The individual bits of tubular peroxisomes drew further apart as the neighboring ER polygons reorganized in different directions (Figure 4D; Movie S6). Observations on the pulling apart and breaking of a tubule through ER reorganization in different directions suggested that the tubular peroxisomes were somehow strongly tethered to the ER and not free to slip out of the ER mesh on their own. A software aided 3D volume rendering was carried out to get more insight into the relationship between the ER and the tubular peroxisomes.

Three-dimensional iso-surface rendering of confocal image stacks showed that peroxisomal tubules are closely inter-twined with tubules of the ER (Figure 5A; Movie S7). Such intertwining probably accounts for their fission as the ER undergoes reorganization in an actin-myosin dependent manner. Their position as embedded tubules in the ER also accounts for their dynamic ER-like behavior. Such coincidental behavior of contiguous ER and tubular peroxisomes suggests that they might have strong adherence to each other. Although the membrane contact sites (MCS) between peroxisomes and the ER are not readily visible in our rendered image the weaving of tubules between ER polygons suggests the possibility of such contact and adhesion sites. However, neither confocal imaging nor the iso-surface rendering suggests luminal connectivity between closed peroxisomal tubules and the ER.

The creation of constructs YFP-PTS1 (Mathur et al., 2002), mEosFP-PTS1 (Sinclair et al., 2009), ss-RFP-HDEL (Sinclair et al., 2009) has been described earlier. The apm1-1 mutant line carrying a GFP-PTS1 has been described (Mano et al., 2004) and was used as provided. Seeds of drp3A-3 (SALK_066958) seeds were obtained from the Arabidopsis Biological Research Center (The Ohio State University, Columbus). Stable transgenic lines of wild type Arabidopsis and the apm1-1 mutant expressing one and two targeted fluorescent proteins were created through the floral dip method (Clough and Bent, 1998), and through crossings.

Arabidopsis plants in sterile culture were grown in petri dishes in an incubator maintained at 21 ± 2°C and a 16/8-h light/dark regime using cool-white light of approximately 80–100μmol m⁻² s⁻². To break dormancy A. thaliana seeds were incubated at 4°C for 48 h Growth medium for A. thaliana WT and mutant seedlings consisted of 1% agar-gelled Murashige and Skoog (1962) basal medium containing Gamborg vitamins (M404; PhytoTechnology labs) supplemented with 3% sucrose (pH 5.8). For obtaining etiolated A. thaliana seedlings, petri dishes were wrapped in two layers of aluminum foil right after the cold treatment and a pinhole created in the foil before placing the plates in an upright position. Seedlings were used between 8 and 11 days after sowing on plates for all experiments.

Plant tissue and seedlings were mounted in tap water on a glass depression slide and placed under a coverslip. For plants expressing the photoconvertible mEosFP-PTS1 protein the photoconversion time was varied according to the brightness of the respective organelles. In general, exposure times for peroxisomes were between 3 and 6 s and resulted in bright red organelles. The light source for photo-conversion was a HBO 100 W/2 Mercury Short Arc lamp and the Leica fluorescence filter set “D” (Excitation filter: 355–425 nm; Dichromatic mirror 455 nm; Suppression filter LP 470 nm). The epi-fluorescence setup consisted of a Leica DM-6000CS microscope. Photo-conversion was performed manually by controlling the diaphragm as described earlier (Mathur et al., 2010). Simultaneous imaging of peroxisomal and ER probes was carried out using a Leica TCS-SP5 confocal laser-scanning unit equipped with a 488 nm argon and a 543 nm helium-neon laser. All images were captured using at a color depth of 24bit RGB.

All images and movies were cropped and processed for brightness/contrast as complete image or stacks using either Adobe Photoshop CS3 (http://www.adobe.com) or the ImageJ distribution Fiji (http://pacific.mpi-cbg.de/wiki/index.php/Fiji). Adobe Photoshop was used for annotation of movies. Imaris software (v. 6.4.0; Bitplane AG) was used to render iso-surface 3 D rendering of ER and peroxisomes from confocal image stacks and x-y-time series. All experiments were carried out at least five times.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.