Plastid stromules: video microscopy of their outgrowth, retraction,
tensioning, anchoring, branching, bridging and tip-shedding
Brian E. S. Gunning*1
Plant Cell Biology Group, Research School of Biological Sciences, Australian National
University, Canberra, Australia
Running Head: B. E. S. Gunning: Video microscopy of plastid stromules
*Correspondence and reprints: Plant Cell Biology Group, Research School of Biological
Sciences, Australian National University, PO Box 475, Canberra, ACT 2601, Australia.
E-mail: [email protected] Internet: www.plantcellbiologyonCD.com
1
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 frameby-frame detail, in accord with early observations.
Keywords: Actin; Chloroplast; Cytoplasmic streaming; Mitochondrion; Stromule; Video-microscopy;
Abbreviations: DIC differential interference contrast microscopy; GFP green fluorescent protein.
Introduction
The name stromule (Köhler and Hanson 2000) conveys two major properties of these enigmatic
tubular extrusions from plastids. They are usually less than 0.5µm in diameter and are up to several
tens of µm long. They possess both of the plastid envelope membranes, lack chlorophyll, but contain
rubisco and probably other components of the stroma phase of the parent plastid (Gray et al. 2001,
Kwok and Hanson 2004b). Their existence and widespread distribution among different categories of
plastid and in different types of cell were established many years ago by means of conventional and
phase-contrast microscopy (reviewed in Gray et al. 2001, Kwok and Hanson 2004b), whereas most
recent studies have used green fluorescent protein (GFP) label and confocal or multi-photon
microscopy. Both eras of research produced intriguing discoveries, for example observations of
stromules releasing mitochondrion-like bodies into the cytoplasm (Wildman et al. 1962), and the
ability of stromules to transport GFP (Köhler et al. 2000) and in certain situations to convey it from
one plastid to another (Gray et al. 2001, Köhler et al. 1997, Kwok and Hanson 2003). To learn more
about the dynamic activities of stromules in live cells, differential interference contrast (DIC) optics
and video recording have been used here to observe their behaviour in the context of other cell
components and phenomena that are not readily visualised by confocal microscopy, at time periods
ranging from fractions of a second up to an hour or so. The images are from a collection of movies of
stromules on chloroplasts, chromoplasts and leucoplasts, 22 of which can be viewed in time-lapse
format elsewhere (Gunning 2004). 107 stills are presented here to add new detail to previous
descriptions, and to describe aspects of stromule behaviour that have not been documented previously.
2
Material and Methods
Plant material
The plant material used for video movie sequences of chloroplast and leucoplast stromule dynamics
included peels of lower epidermis plus green and non-green sub-epidermis from the mid-rib region of
the standard petals and the anther filament of Iris unguicularis Poiret (winter-flowering Iris) flowers,
trichomes cut basally from Lycopersicon esculentum Miller (tomato) and Cucurbita spp. petioles,
hand-cut and sledge microtome sections from Spinacia oleracea L. and Brassica albo-glabra (Chinese
broccoli) leaves (kindly prepared by Dr D. Spencer, CSIRO Division of Plant Industry, Canberra);
Nicotiana tabacum L. and N. plumbaginifolia cells in suspension culture, and whole mounts of the
root-hypocotyl junction of Arabidopsis thaliana L. seedlings. Chromoplast stromules were recorded in
peels from young Physalis alkekengi L. (Chinese lantern) calyx tissue and petal trichomes from
Cucurbita spp.
Microscopy
Samples were mounted in water or “stamen hair buffer” (Cleary et al.1992) and examined using a
Zeiss Axioplan microscope with DIC optics and a 100x Planapochromat objective lens. Movies were
recorded in real time using a Sony DXC327P 3-chip video camera as detector, with its sVHS output
directed to a Sony TRV-900 camera, used simply to record the sequences in digital form. The digital
format allowed the video sequences to be imported directly into a computer using Adobe Premiere ver.
6.5, and the same software was used to prepare time-lapse sequences and to capture selected frames.
Results
The sequences in the Figures have been selected to illustrate multiple aspects of the dynamic
behaviour of stromules, described below under a number of overlapping topic headings.
Outgrowth and retraction of stromules
Sub-epidermal tissue in peels from the proximal midrib region of Iris petals (a choice inspired by
descriptions of stromules in Iris tissues by Faull (1935)) has provided good sequences of stromules
growing and retracting from well-developed chloroplasts, as well as an abundance of stromules on
leucoplasts (not shown here, but see Gunning 2004).
Figure 1 (centre image, at 445 seconds from the start of the sequence) shows a group of chloroplasts
with stromules. Green pigment does not extend into the stromules, in agreement with the confocal
fluorescence literature (Gray et al. 2001, Kwok and Hanson 2004b). The other images in Figure 1
show stages of growth and retraction of one of the stromules, at selected time points over a 14 minute
period. The stromule grew out along a track marked by linear streaming of many mitochondria (e.g.
visible at times 0, 44, 100 seconds). At times the extending tip was led by a linearly-associated
3
mitochondrion (e.g. time 145), a type of association also seen during retraction (570) and outgrowth of
a new stromule (835, the mitochondrion is alongside the oblique line). Growth rates (Fig. 3) varied
widely; initially this stromule extended at 0.23µm s-1, but rates as low as 0.05µm s-1 were also
measured. Outgrowth is punctuated with stops and starts and transient retractions (Fig. 3). When fully
extended the stromule was distinctly thinner than it had been earlier, as if attenuated by stretching
(Fig. 1, time 445). Retraction is usually faster than outgrowth: between times 445 and 570 the rate was
-1.16µm s-1 (Fig. 3). The other stromules seen at time 445 remained nearly static throughout the period
of observation, so they do not behave synchronously in the cell.
When long stromules retract they generally do not disappear immediately into the body of the plastid.
Instead they tend to form a loop at the plastid surface, presumably composed of membrane derived
from the extended state (685, also last five images of Fig. 7). Diffraction effects preclude accurate
measurement of the width of the stromules in these DIC images, but if a low estimate of 0.3µm is
used, the surface area of the fully extended stromule in Figure 1 is about 25µm2, equivalent to about
35% of the surface area of the chloroplast body, i.e. a considerable amount of membrane for synthesis
and disposal. When a new stromule emerged (835-864) it did so from the remnant loop. However new
stromules have also been observed emerging from sites other than remnant loops.
Anchoring and stretching of stromules
The deformable nature of stromule membranes and contents is manifest in several ways, including
bending and stretching movements that result from the mechanical effects of cytoplasmic streaming on
plastids that are anchored by one or more points along their stromules.
For example, the two chloroplasts in Figure 2 are bridged by a stromule; another stromule about 15µm
long extends opposite to the bridge. Another, very short, stromule anchored the right-hand extremity
of the right-hand chloroplast (not visible in the selected frames). The distal 8µm of the longer stromule
(marked by arrowheads) remained fixed in position for nearly an hour of observation (only 10 minutes
shown here), while waves of intense cytoplasmic streaming averaging just over 4µm s-1 impacted the
plastid bodies, stretching and relaxing the intervening unanchored regions of stromule (including the
bridge) as it continually changed directions. In the first and last images in Figure 2 the bridge is so
short as to be barely visible, but in the second and third images it is shown stretched to 3-5µm in
length.
Stromules mostly grow downstream along the direction of cytoplasmic streaming (Fig. 1) but can also
grow upstream (bottom row, Fig. 4). As already shown in Figure 2, they can also become upstream
anchors for plastids, tethering them against even rapid currents of cytoplasmic streaming, like boats at
the end of elastic ropes. Figure 4 gives more detail of the latter arrangement in the case of a 10µm long
stromule. At time 0 the stromule was straight, pointing upstream along a (putative) actin cable. The
plastid was suspended in the stream by its stromule, whose tip remained anchored at the same point.
The second image (5.36 seconds after the first) pinpoints an instant when the tip of the stromule
detached from that anchor point. The sudden release of elastic tension created rapidly undulating
4
waves all along it during the subsequent second (5.44-6.24), indicating that it had been uniformly
stretched from its anchor at the tip. During this brief period the released plastid and its now detensioned stromule flowed downstream along with other organelles for about 8µm. The tip then
established another attachment, whereupon continued pressure on the plastid body exerted by
downstream cytoplasmic flow stretched the stromule once again (7.76). It remained in its new position
until 30.08 seconds, when it again detached, became undulating as before, but this time proceeded to
retract completely into the plastid body (at its side, not at the leading tip) (30.12 - 36.48). A new
stromule then emerged from the tip of the plastid and grew upstream (36.48 - 40.04). The putative
actin cable can be seen in many of the images (arrowheads in 12.32-16.52), extending precisely
beyond the tip of the stromule. Five of the images are at 1 second intervals, with movement of the
circled organelle demonstrating the direction of streaming, at an average rate of 2.7µm s-1.
Figure 7 illustrates stromule branching (see below) as well as stretching. After undergoing branching
(images at times 0-9.2) this stromule became stably attached to a fixed point at the cell surface and
was subjected to alternating phases of stretching and recoil as the flowing cytoplasm exerted pressure
on the free-floating plastid body, resulting in up to two-fold changes in the length of the stromule
(times 90-156). The tip remained fixed in position throughout. At moments of excessive stretching the
stromule became beaded, indicating that the lumenal contents can move along it, in and out of the
beads as they form and disperse (90, 101).
As shown in Figure 2, anchoring of stromules can occur at positions along their length, and not just at
their tips. Figure 7 (101) shows a distinct elbow about half way along a stromule. The streaming
pattern in the cell indicated that the portion of stromule distal to the elbow was stretched along a line
of moving organelles. This particular elbow did not persist for long, but it clearly was a transient
anchor point, with the portion of stromule between the plastid body and the elbow lying free.
Moreover the position of the elbow shifted with time, shortening and lengthening the free portion.
Figure 6 gives an example of more sustained anchoring at two elbows along the length of a 45µm long
stromule. The anchored elbows (vertical arrows) remained at the same positions despite rapid
streaming towards the plastid body. They were not on an obvious actin cable that lay across the field
of view (slanting double-headed arrows), along which organelles were moving rapidly, at times
accumulating like a log jam in the angle between the stromule and the actin cable, causing the
stromule to stretch, flex (38) and recover when the accumulation dispersed (74.6). As in Figure 7 (121467) this stromule terminated at a spherical body. Its other feature of interest is a stable inflated region
half way between the two elbows.
The DIC optical system did not reveal any structural components in the cytoplasm alongside attached
elbows on stromules (Figs 2, 6, 7). However, three different morphologies were seen at anchored tips.
In the first type the stromule tip shows no discernible structural attachment (Figs. 2, 4). In the second
type the stromule terminates in an irregular, flattened lobe shown by critical focussing to be at or very
close to the cell surface (Fig. 1 - asterisks and stromule branch, Fig. 6 and Fig. 7 – times 0-9.2). In the
5
third type the stromule is anchored at a spherical, sometimes dimpled, body about 1.5µm in diameter
(Figs. 6, 7).
Branched and multiple stromules
In Figure 7 the first set of stills (time 0-9.2 seconds) presents the history of a short-lived branch. It
emerged from the terminal lobe of a stromule and grew along the track of a newly arrived stream of
organelles, as if a portion of the parent stromule membrane at the lobe had become attached and then
pulled out. The streaming track shifted leftwards during the few seconds shown, and the branch
followed it, showing that its junction with its parent stromule was plastic. At about 7.6 seconds the
branch detached abruptly from the streaming cytoplasm, whereupon it veered and retracted.
While Figure 7 illustrates translocation of a branch along a stromule, Figure 8 shows that the points of
attachment of stromules to the plastid body can also be mobile. This Figure shows an attachment point
moving over the surface of the plastid to where another stromule was beginning to emerge, and then
back to its original site, all in a period of 16 seconds.
Figure 1 (which also exemplifies multiple stromules per plastid) includes images of a stromule branch
point moving both along a parent stromule and over the plastid body. A branch (horizontal arrows)
with a terminal flattened lobe extended from near the base of the outgrowing stromule in the early part
of the sequence (0-145). Its position in the cell seemed to be fixed, so that when the chloroplast was
pulled in the direction of growth of the main, outgrowing stromule by about 5µm, the branch origin
became shifted back (leftwards in Fig. 1) by that same distance. By then it emerged from the body of
the chloroplast. It persisted there, mostly out of focus (but its position is indicated by short arrows in
other images). A third stromule (asterisks) was in line, opposite to the main one. Initially it was very
short and also had a terminal lobe. It became extended by about 5µm when the chloroplast shifted
away by that distance. In both cases the terminal lobes were fixed in position, as if anchored at or very
close to the cell surface. Yet another, short-lived, stromule grew along an oblique track of streaming
near the end of the sequence (835, oblique arrowhead at tip).
Shedding of a stromule tip body
Starting at 106 seconds in Figure 7, an elongated terminal bead, which had been present for a
considerable time and was unambiguously an integral part of the stromule, metamorphosed in just over
one second into a spherical body which appeared to be demarcated from, but attached to, the stromule
tip (105.72 and subsequent 9 images of the tip, showing times elapsed from 105.72). This 1.5µm
diameter body remained at the tip for >5 minutes despite continual cytoplasmic streaming sweeping
past it. Then, commencing at time 467, the remaining images in Fig. 7 (times elapsed after the 467
second time point are shown) illustrate the sudden shedding of the terminal body (0.2-1.0) and the
instant reaction of the stromule, which recoiled from a straight to an undulating structure that quickly
retracted into a loop of membrane at the chloroplast body (2.2-5).
6
Although several examples of spherical bodies at stromule tips were seen (e.g. Fig. 6), no other
recordings of their release into the streaming cytoplasm were obtained.
Bridging:
Stromules that form bridges between plastids have been observed by confocal microscopy and are the
path of GFP exchanges between plastids (see Introduction). Figures 2 and 7 show plastids that are
linked to one another in this way, though in Figure 7 they are so close that the bridge cannot be seen.
Figure 5 illustrates breaking and re-establishing a bridge. Initially the bridge was thicker than most
stromules (time 0) but it became attenuated to more standard dimensions later (33 seconds), when the
bridge and an unbridged stromule (also in 33) became stretched along a cytoplasmic stream. At 59
seconds the two plastids could be seen to move independently of one another and were completely
separated (59-90). They then rejoined, first by a thick bridge (102) and later became more confluent
(109, 118). By this time they no longer had an anchoring stromule (cf earlier) and moved in tandem
out of the field of view (beginning at 118).
Mitochondrial equivalents of stromules:
Many of the observations described above imply interaction of the external membrane of stromules
with cytoskeletal strands, presumably actin, in accord with the inhibitor studies of Kwok and Hanson
(2003) and double-labelling of actin and stromules (Kwok and Hanson 2004a). It is known that
plastids can be moved around cells by cytoskeletal actin, and it is possible that outgrowth of stromules
represents a special case of this form of interaction, in which molecular binding leads to initiation and
then directed growth along the actin. Mitochondria also move along actin cables, and can become
extremely attenuated prior to fission (Gunning 2004) rather like the stromule in Figure 7 at time 156.
Hence it is relevant that mitochondria too can sometimes be seen to form transient tubular extensions
(Fig. 9). As seen in the case of plastids, this mitochondrial equivalent of a stromule emerged along a
pathway of cytoplasmic streaming that was revealed by the direction of movement of particulate
material.
Discussion
Video-rate microscopy augments views of stromule dynamics derived from confocal fluorescence in
conjunction with DIC imaging (e.g. short time-lapse movies in Kwok and Hanson 2004a). It confirms
spatial associations of stromules and actin, as seen in double-labelling experiments (Kwok and Hanson
2004a), though the bundles that are observed here are identified merely as visible cables, or often just
as tracks of localised cytoplasmic streaming. Stromules, or parts of them, lie and grow along these
tracks, often retracting when they dissociate, consistent with the idea that their outgrowth and
retraction are conditioned by local interactions of individual stromules with cytoskeletal filaments.
7
Whereas outgrowth along actin cables suggests participation of some form of motor protein, anchoring
is indicative of tethering complexes. Anchoring has not figured in discussions of stromule dynamics or
function (Gray et al. 2001, Kwok and Hanson 2004b), although attention has been drawn to the
existence of “contact points” between stromule tips and the inner face of the plasma membrane (Kwok
and Hanson 2004c). Anchoring is conspicuous in some of the cell types examined here, but may not
occur, or would be less obvious, in cells that do not exhibit rapid streaming. It is not confined to the
tip, but can occur at intercalary points (which may appear as persistent or transient sharp elbows) or
along several µms of stromule. Anchored tips often have flattened lobes, plaques or spherical bodies,
but anchored elbows do not. It is likely that the lobes are at the cell surface while the elbows may be
movable attachments to cytoskeletal fibrils (which were not visualised with the optical system used
here). When combined with anchoring or tethering, streaming can stretch stromules, leading to elastic
recoil upon release. Release often leads to retraction of stromules back into loops of membrane at the
plastid body, as seen in many electron micrographs (Gray et al. 2001).
Retraction is usually several times faster than outgrowth (documented in Fig. 3), which occurs at much
the same speed as the mobility of “batches” of GFP along stromules, as detected and quantitated by
photon correlation spectroscopy (Köhler et al. 2000). Indeed bulk movement in the form of growth
could underlie some instances of movement of stromule contents, as assessed by that technique.
However, in chromoplasts with stromules (or spicules of stromule dimensions), pigment particles can
be seen moving longitudinally in the absence of growth, presumably in the stroma compartment
(Gunning 2004), confirming the existence of batch transport. Other clear evidence for mobility of the
contents is seen in the formation and dissipation of swollen beads when stromules are stretched and
relaxed. The stromule beads seen here invariably have been interconnected by very tenuous stretches
of membrane, presumably with minimal stromal contents. As suggested by Pyke and Howells (2002),
this redistribution of contents probably accounts for the appearance of strings of apparently discrete
spindle-shaped vesicles when GFP fluorescence labels the contents but not the outer membranes of
stromules.
Stromules can undergo much longitudinal stretching without rupturing (up to two-fold in the movie of
Fig. 7; even more in the bridge portion of Fig. 2), raising questions about the molecular basis of their
resilience and tethering. The behaviour of branch junctions, which can flow along parent stromules
(Fig. 7) or over the plastid body (Fig. 8), suggests that the membranes are quite fluid. Nevertheless,
markedly stretched parts of anchored stromules, consisting of little other than membrane, can clearly
withstand strong convective forces arising from bulk flow of cytoplasm. Pronounced stretching also
occurs in mitochondria, where it may precede fission (unlike stromules) (Gunning 2004). Indeed the
observation of mitochondrial equivalents of stromules (Fig. 9) suggests one common mode of origin
and outgrowth, when points on the outer membrane of plastids and mitochondria bind to a cytoskeletal
element such as actin to initiate downstream outgrowth of tubules. Stromules in cells with little or no
cytoplasmic streaming may have different modes of origin, including an inherent capacity to grow,
given that stromule formation has been observed on an isolated chloroplast (Spencer and Unt 1965).
8
Three of the Figures – all from tomato trichomes – show pairs of chloroplasts bridged by stromules,
and Figure 5 may well be the first complete visualisation of breaking and making such bridges (for
literature on bridges see Gray et al. 2001, Kwok and Hanson 2004b). In this material the bridges can
expand to a diameter that is large enough to accommodate portions of the thylakoid systems (Fig.5
times 0 and 118), and conversely they can be stretched to more standard stromule diameters (0.3 – 0.5
µm) (Figs.2, 6). Why they should be relatively common in tomato trichomes is not known. Certainly
mere proximity of chloroplasts is not sufficient, for cytoplasmic streaming frequently brings them into
contact without bridge formation. Under certain stress conditions plant mitochondria can form a
reticulum (Van Gestel and Verbelen 2002), or a reticulum can be a natural state (Gunning 2004), and
the ability of plastids to bridge to one another may involve comparable recognition and fusion
processes.
The question of whether stromules can release mitochondrion-like particles has been debated (Gray et
al. 2001) since this was first seen in movies made using phase-contrast microscopy (Wildman et al.
1962). Close associations of mitochondria and stromules are very common, as observed in almost all
of the examples assembled here, but the present movies give the strong impression that the great
majority of the associations arise fortuitously, either because the two are interacting with and moving
along the same actin bundle (e.g. Fig. 1, 2, 4), or because cytoplasmic streaming causes mitochondria
(etc) to pile up against anchored stromules when they obstruct free passage of organelles (Fig. 6).
Prolonged spatially-specific associations have not been detected, other than at the tips of stromules
(e.g. Fig. 1), which evidently can have binding properties, as yet uncharacterised. Against this
background the formation and subsequent dissociation of a terminal spherical body illustrated in
frame-by-frame detail in Figure 7 is unequivocal but also a rare event. Although this “budding” could
be an example of a role for stromules in contributing free organelles with particular function(s) to the
population in the cytoplasm, it is more likely to be a case of a stromule shedding a terminal attachment
region prior to retraction. As shown in Figure 7 (time 467 et seq.) it would not be possible to follow
the fate of these released particles for very long unless specific labelling procedures are used.
Nevertheless, the phenomenon could account for sporadic observations of apparently free fluorescent
objects in cells expressing GFP targeted to the plastid stroma (Arimura et al. 2001).
Acknowledgment
I thank Don Spencer for many helpful discussions and information on his pioneering work on
stromules in collaboration with S. Wildman, and for preparing sections of leaf material.
9
References
Arimura S-I. Hirai A, Tsutsumi N (2001) Numerous and highly developed tubular projections from
plastids observed in Tobacco epidermal cells. Plant Science 160: 449-454
Cleary AL, Gunning BES, Wasteneys GO, Hepler PK (1992) Microtubule and F-actin dynamics at the
division site in living Tradescantia stamen hair cells. J Cell Sci 103: 977-988
Faull AF (1935) Elaioplasts in Iris: a morphological study. J. Arnold Arboretum 16: 225-267
Gray JC, Sullivan JA, Hibberd JM, Hanson MR (2001) Stromules: mobile protrusions and
interconnections between plastids. Plant Biology 3: 223-233
Gunning BES (2004) Plant Cell Biology on CD, information for students and a resource for teachers.
BESGunning Pty, Canberra http://www.plantcellbiologyonCD.com
Köhler RH, Cao J, Zipfel WR, Webb WW, Hanson MR (1997) Exchange of protein molecules
through connections between higher plant plastids. Science 276: 2039-2042
Köhler RH, Hanson MR (2000) Plastid tubules of higher plants are tissue-specific and
developmentally regulated. J Cell Sci 113: 81-89
Köhler RH, Schwille P, Webb WW, Hanson MR (2000) Active protein transport through plastid
tubules: velocity quantified by fluorescence correlation spectroscopy. J Cell Sci 113: 3921-3930
Kwok E, Hanson MR (2003) Microfilaments and microtubules control the morphology and movement
of non-green plastids and stromules in Nicotiana tabacum. The Plant Journal 35: 16-26
Kwok E, Hanson MR (2004a) In vivo analysis of interactions between GFP-labeled microfilaments
and plastid stromules. BMC Plant Biology 2004, 4: 2 http://www.biomedcentral.com/1471-2229/4/2
Kwok E, Hanson MR (2004b) Stromules and the dynamic nature of plastid morphology. J Microscopy
214: 124-137
Kwok E, Hanson MR (2004c) Plastids and stromules interact with the nucleus and cell membrane in
vascular plants. Plant Cell Reports (in press)
Pyke KA, Howells CA (2002) Plastid and stromule morphogenesis in tomato. Annals of Botany 90:
559-566
Spencer D, Unt H (1965) Biochemical and structural correlations in isolated spinach chloroplasts
under isotonic and hypotonic conditions. Aust J Biol Sci 18: 197-210
Van Gestel K, Verbelen J-P (2002) Giant mitochondria are a response to low oxygen pressure in cells
of tobacco (Nicotiana tabacum L.). J Exp Bot 53: 1215-1218
Wildman SG, Hongladarom T, Honda SI (1962) Chloroplasts and mitochondria in living plant cells:
cinematographic studies. Science 138: 434-436
10
Figure Legends:
Fig. 1. Growth and retraction of stromules on Iris unguicularia chloroplasts. Elapsed times (seconds)
are shown. Time 445 shows stromules on three chloroplasts, with a nucleus in the background.
Vertical arrows mark a (presumed) actin cable along which organelles were streaming. Two of the
stromules are partially aligned along it. The remaining images depict growth and retraction of the
lower stromule, which also lay along a track of cytoplasmic streaming. Stromule tips are marked by
arrowheads in each image. At times 0-145 mitochondria were moving along the same track and at 145
and 570 (during retraction) mitochondria associated end to end with the stromule tip (ellipses). They
are distinctly wider than the stromule. This chloroplast possessed multiple stromules. In addition to the
main one there was a short stromule (asterisk) pointing in the opposite direction, and a branch
(horizontal arrows). Both of these had terminal lobes, flattened close to the cell surface. During
extension of the long stromule, the chloroplast was pulled to the right by about 5µm. Concomitantly
the oppositely-directed stromule became elongated by the same amount while its terminal lobe
remained in place, as if firmly anchored. Similarly the point of origin of the branch became pulled to
the left (relative to the rightward-moving chloroplast body), suggesting that the terminal lobe on this
stromule was also anchored: its relative movement resulted in the branch origin shifting from the main
stromule (time 0) to the plastid body (445 and subsequent).
Fig. 2. Tethering and bridging by stromules in a tomato trichome. Times are in minutes:seconds in
this sequence, recording conformational changes in a pair of chloroplasts that were bridged by a
stromule. At times they were isolated and quiescent, at times they were washed by cytoplasm moving
leftwards, at times rightwards, and at times they were at the interface between oppositely directed
flows (directions of major flows at the time points shown are indicated by arrows; particle movement
was measured at just over 4µm s-1). During 60 minutes of recording the distal part of the long stromule
(between arrowheads) and the tip of a very short (barely visible) stromule at the right pole of the
terminal chloroplast remained anchored in precisely the same positions, while the intervening regions
of the terminal and bridging stromules bent, stretched and recovered according to the directions of ebb
and flow of the streaming cytoplasm.
Fig. 3. Stromule outgrowth and retraction: the graph at the left side presents data for the main
stromule in Figure 1; the right-hand graph represents another stromule in the same Iris unguicularia
petal tissue (not illustrated).
Fig. 4. Tethering and release of tension upon detachment. These stills start with a stromule attached at
its tip (down arrowheads in all images), possibly to a presumed actin cable that can be seen running
past the tip (visible in many images and marked by up-arrowheads at times 12.32 - 16.52 seconds).
The straight stromule at time 0 was under tension and the next 7 stills show its recoil when the tip
11
detached, allowing the chloroplast to drift in the streaming cytoplasm. At time 7.76 the tip reestablished an attachment and the stromule became straight and stretched once again. The new
attachment held for 23 seconds against the bulk movement of cytoplasm (which is illustrated by
leftward movement of the circled particle at an average velocity of 4.8µm s-1). The final frames show a
second detachment at time 30.08, followed by elastic recoil as before, but then proceeding to complete
retraction and the start of outgrowth of a new stromule, still against the direction of streaming. The
images down to 31 seconds are in register, showing the stage when the chloroplast was anchored by its
stromule, which stretched slightly (7.76-30.08) and the stages when stromule detachment allowed the
chloroplast to drift freely (5.36-7.76 and 30.12-31.68).
Fig. 5. Breaking and reinstating a bridge. At time 0, two chloroplasts in a tomato trichome are joined
by a connection that is wider than the normal stromule diameter. The connection narrows (33
seconds), the chloroplasts separate and move independently (49-90), rejoin (102-109) and drift away
in unison (118). At times 0 and 14 a chloroplast with a stromule lies below the bridged pair (out of
focus at time 0), marking a pathway of streaming cytoplasm that is later joined by stromules
emanating both from the bridged chloroplasts (33, 49) and the separated chloroplasts (59, 77).
Fig. 6. Intercalary anchor points. Three stages (times in seconds) of flexing and recovery of a 45µm
long stromule on a tomato trichome chloroplast. The stromule was anchored at two static elbows along
its length (vertical arrows). As the cytoplasm streamed leftwards, organelles often piled up in the angle
between the stromule and a prominent actin (presumed) cable (sloping double-headed arrows). Note
that in contrast to Figure 1, in this case the stromule and the cable shift in their relative positions, so
they appear not to be tethered to each other, though they cross twice.
Fig. 7. Branching, stretching, retraction and apical shedding in a stromule emanating from a linked
pair of plastids in a tomato trichome cell. The first 11 views show the origin, outgrowth and retraction
of a short branch. It starts at a terminal lobe of the main stromule (time 0) and grew along a track of
cytoplasmic streaming before detaching at time 7.56, whereupon it withdrew. The tip of the main
stromule stayed at the same position in the cell for nearly 8 minutes. The next four images (times 90 156) illustrate stages in stretching (with lumenal contents becoming squeezed into “beads”
interconnected by tracts of narrow membrane) and recoil as the plastids were intermittently pulled
along in the streaming cytoplasm. Stage 101 illustrates a sharp elbow which moved rapidly along the
stromule, just as a bend can be moved along a piece of stretched string that is tethered at both ends.
During this period an elongated bead at the tip of the stromule rapidly changed shape into a terminal
spherical body. The metamorphosis is shown frame-by-frame starting at 105.72 (with elapsed times
thereafter in the 10 insets of the tip region). The spherical tip body, now indicated by horizontal
arrows, remained at the stromule tip for several minutes until it dissociated at 467 seconds. Its
movement during the next 2.6 seconds is shown in the next 6 images. After separation it soon became
12
indistinguishable (using DIC) from other organelles (e.g. stage 2.2). Its shedding at time 467 was
followed immediately (remaining times shown are elapsed from 467) by the start of complete
retraction of the stromule into a loop of membrane at one pole of the parent chloroplast (last 5 images).
Fig. 8.
Mobility of a stromule attachment point: images illustrating movement of the point of
emergence of a stromule from a plastid body during a 16 second period (tomato trichome). The
attachment point (arrowheads) migrates over the plastid body and back again.
Fig. 9. Mitochondrial equivalents of stromules. A mitochondrion in a Brassica mesophyll cell (first
image) interacted with a path of streaming cytoplasm (not visible in the stills) to develop a stromulelike protrusion (0-6 seconds), then retracted it (10-16) and started to produce another (20) along a
different stream.
13