Download Plastid and Stromule Morphogenesis in Tomato

Annals of Botany 90: 559±566, 2002
doi:10.1093/aob/mcf235, available online at www.aob.oupjournals.org
Plastid and Stromule Morphogenesis in Tomato
K E V I N A . P Y K E * and C A R O L I N E A . H O W E L L S
Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington campus, Loughborough,
Leicestershire LE12 5RD, UK
Received: 23 May 2002 Returned for revision: 16 July 2002 Accepted: 26 July 2002 Published electronically: 2 October 2002
By using green ¯uorescent protein targeted to the plastid organelle in tomato (Lycopersicon esculentum Mill.),
the morphology of plastids and their associated stromules in epidermal cells and trichomes from stems and
petioles and in the chromoplasts of pericarp cells in the tomato fruit has been revealed. A novel characteristic of
tomato stromules is the presence of extensive bead-like structures along the stromules that are often observed as
free vesicles, distinct from and apparently unconnected to the plastid body. Interconnections between the red
pigmented chromoplast bodies are common in fruit pericarp cells suggesting that chromoplasts could form a
complex network in this cell type. The potential implications for carotenoid biosynthesis in tomato fruit and for
vesicles originating from beaded stromules as a secretory mechanism for plastids in glandular trichomes of
tomato is discussed.
ã 2002 Annals of Botany Company
Key words: Plastid morphogenesis, chromoplast, stromule, tomato.
INTRODUCTION
Plastids are a family of cellular organelles that comprise a
variety of different types found in different sorts of plant
cell. Plastids are normally differentiated in speci®c cell
types in relation to cellular function. Although green
chloroplasts in leaf mesophyll cells are the best studied
plastid type, a wide variety of other `non-green' plastids
exists in other cells within the plant which carry out a wide
variety of cellular functions critical to cellular integrity. An
understanding of the developmental cell biology of nongreen plastids has been limited by dif®culties in resolving
the structure, morphology and population dynamics due to a
lack of natural pigmentation, making visualization dif®cult.
Recently, green ¯uorescent protein (GFP) has been used to
visualize several different non-pigmented cellular organelles including nuclei (Chytilova et al., 1999) and mitochondria (KoÈhler et al., 1997a). The application of GFP
technology to plastid development has been particularly
successful in revealing the structure of chloroplasts and
some non-green plastid types. These studies have used
either GFP targeted to the plastid (KoÈhler et al., 1997b;
KoÈhler and Hanson, 2000), or GFP expressed transplastomically from a transgene residing on the chloroplast genome
(Gray et al., 1999; Shiina et al., 2000). Using either strategy,
thin ®lamentous structures termed stromules have been
identi®ed emanating from chloroplasts and some other
plastid types lacking substantial chlorophyll, and evidence
suggests that GFP protein molecules can pass between
plastids (KoÈhler et al., 1997b; Hibberd et al., 1998; KoÈhler
and Hanson, 2000; Gray et al., 2001). At present the
function of stromules in plastid biology is unclear. To date,
observations of stromules have been made principally in
* For correspondence. Fax + 44 (0)115 9513298, e-mail kevin.pyke@
nottingham.ac.uk
cells of tobacco and arabidopsis (Tirlapur et al., 1999), and
thus the range of species and plastid types in which these
structures have been partially characterized is limited.
A plastid type of major importance in fruit development
is the chromoplast which has been studied extensively at the
molecular and biochemical level (Camara et al., 1995) in
several ripening fruit systems, including Capsicum species
(Hugueney et al., 1995) and tomato (Fraser et al., 1994), as
well as in ¯ower petals (Vainstein et al., 1994).
Chromoplasts contain novel pigments that confer colour
on fruits. In tomato, the red coloration of the ripe fruit
results primarily from the production of two carotenoid
compounds: lycopene and b-carotene. The biochemical
synthetic pathways of both of these pigments and the
enzymes involved have been characterized in detail, mainly
in tomato (Cunningham and Gantt, 1998). These particular
carotenoids have also been highlighted as compounds that
provide health bene®ts when present in food, and there is
thus signi®cant interest in increasing their levels in tomato
fruit (Romer et al., 2000). Although many studies have
analysed the mechanism by which these carotenoids are
synthesized in chromoplasts (Camara et al., 1995), an
understanding of the cell biology of the chromoplast
organelle in which they are synthesized and accumulate is
very poor. Chromoplasts have been studied primarily by
electron microscopy, and the accumulation of b-carotene in
vesicles within the chromoplasts and the synthesis of
lycopene in crystalline bodies have been described
(Bathgate et al., 1985; Thelander et al., 1986). However,
little is known of the developmental cell biology processes
that occur as green chloroplasts in unripe fruit differentiate
into chromoplasts in ripe red fruit. Consequently, the main
aim of this study was to exploit GFP targeted to plastids in
tomato to determine the extent and complexity of
chromoplast structure in tomato fruits and to compare
ã 2002 Annals of Botany Company
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Pyke and Howells Ð Plastid and Stromule Morphogenesis in Tomato
chromoplast morphogenesis with that of plastids in other
cells types within the tomato plant. In addition, we wished
to determine the extent to which stromules are an important
morphogenic feature of chromoplast structure in ripe tomato
fruit.
MATERIALS AND METHODS
Plant material
Transgenic plants of tomato (Lycopersicon esculentum Mill.
`Ailsa Craig') containing a plastid-targeted GFP construct
were generated by transforming tomato stem explants and
selecting antibiotic resistant transformants on kanamycin
(50 mg ml±1) and carbenicillin (250 mg ml±1) using
previously described methods (Bird et al., 1988). The
GFP targeting construct was a gift of Professor Maureen
Hanson (Cornell University, Ithaca, NY, USA) and consists
of a GFP4 coding region fused with a RecA plastid transit
sequence driven by a double 35S CaMV promoter (Kohler
et al., 1997b). Seed from selected T0 plants was collected
and sown under glasshouse conditions with supplementary
lighting, and plants showing superior GFP ¯uorescence in
plastids were identi®ed by screening.
Tissue sampling and microscopic imaging
To observe plastids exhibiting GFP ¯uorescence in
epidermal and trichome hair cells, strips of epidermis
containing trichomes were peeled from stems and leaf
petioles of mature transgenic tomato plants and mounted on
glass slides in Vectashield (Vector Laboratories,
Peterborough, UK) to reduce ¯uorescence fading. Pieces
of pericarp tissue were cut from whole tomato fruit and
mounted on slides in a similar manner. Samples were
observed using a Nikon Optiphot ¯uorescence microscope
using a DM 510 ¯uorescence ®lter block and images were
obtained either by photomicrography on Fuji 400 ASA ®lm,
or by using a Basler A113CP digital camera linked to a
Lucia Image analysis system (Nikon). Images were
montaged using Adobe Photoshop. Such images show
¯uorescent plastid bodies that contain varying levels of
chlorophyll as a combination of red chlorophyll and green
GFP ¯uorescence. Confocal imaging was carried out using a
Leica TCS-4D confocal microscope with an Argon Krypton
laser and a Leica DMRBE ¯uorescence microscope. GFP
and chlorophyll were imaged using 488 nm and 568 nm
laser lines, respectively. Images were displayed as
maximum intensity projections of optical slices using the
Scanware software provided.
Protoplast preparation
Protoplasts from mature tomato fruit were made using the
protocol of Lindsay and Wei (1999). Tissue from whole
segments of ripe tomato fruit was incubated in 0´5 M
mannitol at 25 °C for 60 min. This was then replaced by
an enzyme digest solution containing 1 % (w/v) cellulase,
0´25 % (w/v) Macerozyme, 8 mM CaCl2 and 0´5 M mannitol.
Fruit tissue was digested overnight in the dark at 22 °C. A
Pasteur pipette was used to gently suck up and down the
digested material to release protoplasts into the media. The
solution was then ®ltered through a 100-mm sieve followed
by a 50-mm sieve, and the ®ltrate was centrifuged at 60 g for
5 min. The pellet was gently resuspended in buffer A
containing 0´33 M sorbitol, 50 mM Hepes-NaOH (pH 6´8),
2 mM Na2EDTA, 1 mM MgCl2 and 1 mM MnCl2 (Robinson,
1985) prior to being recentrifuged under the same conditions and resuspended in buffer A. The yield of protoplasts
using this method was relatively low, principally because
fruit pericarp cells are very large and easily broken, and also
because the jelly tissue adjacent to the seeds in the tomato
fruit contains partially degraded cells normally.
RESULTS
Epidermal cell plastid morphogenesis
Epidermal plastids in tomato contain low levels of chlorophyll and commonly have central constrictions (Fig. 1A)
suggesting that a signi®cant proportion is in the process of
plastid division. However, plastid numbers remain low in
these cells and such divisions rarely go to completion (data
not shown). Stromule-like features revealed by ¯uorescence
of transgenically targeted GFP are rare in epidermal plastids
although they are more commonly observed than in
chloroplasts of tomato leaf mesophyll cells where stromules
are rarely observed (data not shown). Occasional epidermal
plastids are seen to possess short stromules (Fig. 1B), which
are thin tubular structures with little apparent structure and
which can be present on both dividing and non-dividing
plastids (Fig. 1B). However, a few epidermal plastids
possess longer, more complex stromule-like features
(Fig. 1C), which are highly structured and have a beadlike appearance where packets of GFP are visible approximately equally spaced along a strand which itself is barely
F I G . 1. Plastids from stem epidermal cells of tomato plants visualized with plastid-targeted green ¯uorescent protein. A, Epidermal cell plastids in
division showing central constriction, but lacking stromules. B, Short simple stromules emanating from epidermal cell plastids (left hand plastid in
division). C, A dividing epidermal cell plastid with a long thin stromule showing distinct variation in GFP ¯uorescence along its length. Bars = 1 mm.
Pyke and Howells Ð Plastid and Stromule Morphogenesis in Tomato
561
visible (Fig. 1C). Even so, stromules are rare features of
epidermal plastids in tomato.
Trichome plastid morphogenesis
Trichome cells provide an ideal cell type in which to view
plastid morphology as revealed by GFP ¯uorescence in
fresh tissue since they are external to the plant and easily
viewed in fresh epidermal peels. Two types of glandular
trichome are present in the tomato epidermis (McCaskill
and Croteau, 1999): pointed trichomes and those with a
globular structure at the tip consisting of four cells. In both
cases the trichome stalk is multicellular. Plastid morphogenesis in stalk cells of the two types of trichome from both
the stem and leaf petiole is highly variable, and a variety of
different plastid morphologies can be observed in the same
stalk cell. However, no speci®c characteristic of trichome
plastid structure was observed that was related to a speci®c
trichome type. Some trichome plastids possess relatively
simple stromules showing variations in GFP density along
the length (Fig. 2A), whereas others possess shorter thicker
stromules that show architectural features such as bifurcation (Fig. 2B) or fusing of stromules to each other or the host
plastid body (Fig. 2C) suggesting that such structures could
surround adjacent unseen organelles. A more commonly
observed type of morphology in tomato trichomes is that of
a highly irregular cluster of plastid body parts (Fig. 2D and
E) which, at the most extreme, are held together in a chain
by thin stromule-like features. In such morphologies it is
dif®cult to differentiate between the main plastid body and
beaded structures on stromules, or to be sure if the major
plastid body parts were originally separate plastids that have
joined or are parts of the same original plastid. In some
cases spherical ring structures can form at the end of the
chain (Fig. 2D). Such morphology of tomato trichome
plastids was unexpected and suggests that the distinct beadlike structures on stromules may be involved in a continuum
of change in the morphology of the plastid. Although
structures such as those shown in Fig. 2E were observed to
move as a chain within the cell, movement of the bead-like
structures along stromules has not been observed.
Occasionally two obviously distinct plastids are clearly
joined by a stromule with other adjacent stromules showing
extensive bead-like structures that appear to lack linking
stromules (Fig. 2F) and resemble a bunch of grapes. Such
structures appear to be retracted beaded stromules which,
when extended, appear as in Fig. 2E. Confocal imaging of
chlorophyll and GFP separately in tomato trichome plastids
further revealed that, whereas chlorophyll is restricted to the
main parts of the plastid body (Fig. 3A), GFP ¯uorescence is
also observed in distinct round bead-like structures (Fig. 3B)
which appear to originate from the plastid body but are not
visibly connected to it. Although it is not possible to
determine if these small vesicle-like structures remain
attached to the plastid body by a stromule lacking chlorophyll and GFP, it would seem possible that these structures
may represent vesicles arising from trichome plastids that
move into the surrounding cytosol containing stromal
components but not chlorophyll. Since a major function of
these trichomes is secretion, in which plastid metabolism is
F I G . 2. Plastid morphogenesis in trichome hair cells from the stem and
petiole of tomato plants. A, Single long thin stromule showing variation
in GFP signal along its length emanating from a single trichome plastid.
B and C, Short simple stromules emanating from plastids and showing
ring-like structures formed by stromule bifurcation (B) and stromule
fusion (C) either between two stromules or with the host plastid. D,
Highly complex plastid morphology revealing a multi-parted plastid body
and with distinct terminal ring-like structures. E, An elongate plastid
composed of two major body parts joined by thin stromule-like structures
decorated by extensive beading of variable size. F, Two trichome plastids
joined by a stromule with other adjacent stromules showing extensive
bead-like structures. Bars = 1 mm.
involved, we suggest that these could function in this
process.
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Pyke and Howells Ð Plastid and Stromule Morphogenesis in Tomato
Chromoplast morphogenesis
Ripening of tomato fruit involves the differentiation of
chloroplasts in young green fruit into chromoplasts in
mature ripe red fruit. The major plastid-containing cells of
the tomato fruit are pericarp cells, which are large
vacuolated cells that form a layer several millimetres thick
immediately internal to the epidermis of the fruit. In green
fruit these pericarp cells are full of chloroplasts. Searches
for stromule-like projections in chloroplasts of green tomato
fruit failed to reveal such structures in these cells.
Chromoplasts are revealed within the pericarp cells of the
ripe tomato fruit as large populations of small red pigmented
F I G . 3. Confocal imaging of chlorophyll ¯uorescence and GFP
¯uorescence in plastids from trichome hair cells from the stem of tomato
plants. Chlorophyll ¯uorescence (A and C) and GFP ¯uorescence (B and
D) are shown for the same plastids. GFP is observed in the main plastid
body in which chlorophyll resides but also in distinct round vesicle-like
structures which are associated with the main plastid body and do not
contain chlorophyll. Images are maximum projections of 36 confocal
images over 3 mm of the z-axis. Bar = 5 mm.
bodies (Fig. 4A) which can reach populations of between
500±1000 per pericarp cell (data not shown). Chromoplasts
are normally distributed throughout the pericarp cell but are
often localized in clusters (Fig. 4B). Plastid-targeted GFP
co-localizes with the red chromoplast bodies in these
pericarp cells (Fig. 4C). The red carotenoid lycopene is
deposited in crystalline form within the chromoplast and
such elongated red lycopene crystals often deform the
chromoplast membrane dramatically. Figure 5A shows a
long thin red lycopene crystal emanating from the main
chromoplast body, and the targeted GFP ¯uorescence
(Fig. 5B) shows the same pattern, providing evidence that
such crystals remain bounded by the chromoplast envelope
membranes. Lycopene itself does not show ¯uorescence
under these conditions in control plants. GFP ¯uorescence
within the chromoplast membrane revealed a complex
system of stromule-like structures emanating from chromoplast bodies exhibiting extensive architecture and structure.
The simplest stromule structures consist of a short chain of
beads linked together and emanating from the main pigment
body and extending into the cytosol (Fig. 5D). Some
branching in these chains is sometimes observed (Fig. 5D).
It is just possible to distinguish these beaded structures in
bright®eld images in the cytosol (equivalent positions
indicated by arrows in Fig. 5C and D), but without GFP
¯uorescence it would not be possible to associate these with
the red chromoplast body. Often several red chromoplast
bodies are clustered together and emanate a single beaded
stromule as in Fig. 5D. In some cases, GFP ¯uorescence can
be observed in a larger membranous sac around the red
pigmented body within the chromoplast and can also be
associated with chains of larger bead-like structures
containing GFP (Fig. 5E and F) which are not obviously
linked together. In Fig. 5E, the outline of this larger
membrane surrounding the red pigmented body can be seen
in the bright®eld image.
In the majority of chromoplasts within fruit pericarp cells,
a more extensive beaded stromule system is observed
(Fig. 6A±F). In these cases, stromules are long and are
decorated with extensive beading that varies in size and
density, and branching of the stromule system is also
observed. Most signi®cantly, these stromule systems join
adjacent red pigmented chromoplast bodies together to form
a network. Figure 6C and D shows stromules from a single
chromoplast linking two other chromoplast bodies together.
F I G . 4. Pericarp cells and chromoplasts of ripe tomato fruit. A, Single large pericarp cell showing populations of small dark red chromoplasts shown
enlarged in B. C, Fluorescent image of the pericarp chromoplasts shown in B with green ¯uorescent protein targeted to the chromoplast. Bars = 10 mm
(A), 5 mm (B and C).
Pyke and Howells Ð Plastid and Stromule Morphogenesis in Tomato
F I G . 5. Chromoplast morphogenesis in tomato fruit pericarp cells. A and
B, Red chromoplast body with a long straight red pigmented lycopene
crystal to which GFP co-localizes (B). C and D, A long beaded stromule
emanating from a cluster of red pigmented chromoplast bodies which are
just visible in the bright®eld image (C). Equivalent positions on the two
images are arrowed. E and F, Membranes around a red pigmented
chromoplast body revealed by GFP ¯uorescence, which is also visible in
bright®eld (E). Chains of distinct GFP-containing vesicles are associated
with this membranous system. Bar = 500 nm.
F I G . 6. Chromoplast morphogenesis revealed by GFP targeting.
Bright®eld images of red chromoplast bodies (A, C, E and G) are shown
adjacent to the GFP ¯uorescent image (B, D, F and H) of the same ®eld
of view. Extensive stromule networks emanate from the red chromoplast
body which often connects adjacent chromoplasts together (C, D, E and
F). All stromules exhibit extensive architecture with bead-like structures
of varying sizes distributed randomly along the length of the stromule
length. G and H, A protoplast derived from tomato pericarp cells
containing two groups of red pigmented chromoplast bodies which are
interlinked by stromule networking as revealed by GFP ¯uorescence (H).
Bar = 500 nm.
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Pyke and Howells Ð Plastid and Stromule Morphogenesis in Tomato
red chromoplast bodies which are interlinked by extensive
stromule structures with evidence of complex architecture
and beading (Fig. 6G). This suggests that such stromulebased interlinkages between chromoplast bodies are stable
during cell wall degradation and subsequent perturbation in
cytoskeletal architecture, and/or have the capacity to reform
rapidly between adjacent chromoplast clusters. In such
preparations isolated pieces of stromule were also observed
showing clear GFP ¯uorescence (Fig. 7) in which the
membranous tubule is just visible under bright®eld illumination. Such isolated stromules, which are free from the
cellular environment, rarely had extensive architecture, with
only the occasional small bead, and generally appeared as
long, very straight structures.
DISCUSSION
F I G . 7. Isolated stromules from broken tomato pericarp protoplasts.
Isolated pieces of stromule are observed as unpigmented membranous
tubules in bright®eld (A and C) which exhibit GFP ¯uorescence (B and
D). Bar = 250 nm.
In Fig. 6E and F an extensive beaded stromule network joins
two chromoplast bodies together with several junctions
between the two. Such stromule networks are very dif®cult
to observe in bright®eld imaging without GFP ¯uorescence.
Thus, the use of GFP within chromoplasts has revealed that
chromoplast structure within the tomato pericarp cell is far
more extensive than simply the distribution of red
pigmented lumps which are viewed in conventional bright®eld images. Although the bead-like structures on stromules
are reminiscent of vesicular traf®cking along ®laments, we
did not observe movement of these structures in a time-span
of several minutes, although entire stromule structures are
often observed to move around as a chain in the cytosol.
To determine the robustness of these interlinked chromoplasts, protoplasts were made from ripe pericarp fruit
tissue and examined for chromoplast-associated GFP ¯uorescence. Figure 6G and H shows a small protoplast derived
from part of a larger pericarp cell containing two groups of
By utilizing GFP as an organelle-directed marker, the highly
irregular morphology of plastids in tomato trichome cells
and in chromoplasts of the tomato fruit has been revealed
for the ®rst time. A clear difference in plastid morphology
between different cell types has been demonstrated in that
the extent of stromule structures is greatest in chromoplasts
of the tomato fruit pericarp, less extensive in trichome cells,
and minor in epidermal and mesophyll cells. The thin
membranous tubule-like structures termed stromules which
have been observed to emanate from chloroplasts (KoÈhler
et al., 1997b; Gray et al., 1999) and root plastids (KoÈhler
and Hanson, 2000) in tobacco and arabidopsis appear to be
relatively simple structures with little discernable internal
architecture, but which can extend and retract in the
cytoplasm and can form a highly branched system within
cultured tobacco suspension cells (KoÈhler and Hanson,
2000). By extending stromule biology to a new species, it
has been shown that stromules in tomato plastids have a
very distinctive beaded structure that is very different to that
shown previously in tobacco and arabidopsis, suggesting
that stromules could have differing functions in different
cell types and species. Stromules seen emanating from the
red bodies of tomato chromoplasts are long, thin (<100 nm
wide) and highly structured, whereas those in trichomes
appear shorter but are also highly structured, and both show
complex associations between a thin stromule and various
bead-like structures of varying size. Although many of the
larger bead-like structures appear distinct and free from the
plastid body, it is possible that they still maintain contact
with the main plastid body via a thin stromule that lacks
GFP and is thus not visible. Individual GFP-containing
beads distant from the plastid body and with no apparent
connection to it have been observed. The beaded nature of
tomato stromules is highly reminiscent of vesicles traf®cking along a ®lamentous network in which vesicles sometimes move off as free entities. Although clear linkages
between adjacent plastids in both trichomes and fruit
pericarp cells have been demonstrated, rapid movement of
these bead-like structures along stromules has not been
observed. The present observations would suggest that the
short beaded stromules observed in some chromoplasts,
which appear very much like a string of beads (Fig 5), are
the progenitors of much longer beaded stromules in
Pyke and Howells Ð Plastid and Stromule Morphogenesis in Tomato
complex networks between adjacent chromoplasts and in
which the bead-like structures can increase in size. The
relative lack of beaded structures on isolated pieces of
stromule from chromoplasts also suggests that the beaded
architecture of stromules may be stabilized by a cytosolic
component that is lost on isolation. However, the fact that
isolated lengths of stromule can be observed in which GFP
is maintained and seen as a distinct object in bright®eld
suggests that stromule structure itself is relatively stable,
raising the possibility of bulk stromule isolation and
analysis. The presence of beaded stromules in tomato cells
does appear to be a major characteristic of stromule
structure in this species, although it remains to be seen
whether speci®c differences in stromule morphology are
observed across a wider range of species than have been
currently analysed. The width of stromules associated with
chromoplasts is generally less than 100 nm, which is thinner
than previously reported chloroplast stromules (350±850 nm
wide: KoÈhler et al., 1997b; Gray et al., 1999; Tirlapur et al.,
1999). Presumably stromules interconnecting beads but
which lack GFP are even thinner. It is possible that the beadlike vesicles, which are wider than the basic stromule, are
necessary if molecules are to be transported ef®ciently
through such thin stromule networks.
Given the interest in carotenoid pigment biosynthesis in
tomato and efforts to increase carotenoid levels within the
tomato fruit, it is surprising that a detailed understanding of
the morphogenesis of chromoplasts and their proliferation
in pericarp cells is lacking. Previous studies of chromoplasts
in fruits and ¯ower petals (Harris and Spurr, 1969a, b;
Thomson and Whatley, 1980; Whatley and Whatley, 1987;
Cheung et al., 1993) have used electron microscopy
exclusively and, although they have revealed elegant details
of internal chromoplast morphology and of pigment deposition in often irregularly shaped chromoplast bodies
(Whatley and Whatley, 1987), they have failed to reveal
the stromule-like structures or associated vesicles observed
in this study. The discovery of networking between the main
pigmented bodies of chromoplasts also raises the possibility
that precursors in the carotenoid biogenesis pathway could
be moved around the cell between individual chromoplasts
by this network and thus it may be more useful to consider
the red pigment deposits observed in tomato pericarp cells
as all existing within a highly complex chromoplast
organelle.
In contrast to chromoplasts, however, oddly shaped
plastids in trichome cells have been reported previously in
the glandular trichomes of Cannabis plants (Kim and
Mahlberg, 1977) and a role for plastids as the source of
glandular secretions has been implicated in glandular
trichomes of both cannabis and tobacco (Kim and
Mahlberg, 1977; Nielsen et al., 1991). Furthermore,
trichome plastids are essential organelles in providing
metabolic precursors of many secreted compounds, including terpenoid-related molecules (Cheniclet and Carde,
1985; McCaskill and Croteau, 1995).
The demonstration of distinct vesicle-structures associated with stromules and larger, apparently unconnected,
vesicle-like features associated with plastids in both
trichomes and pericarp cells raises the possibility that
565
these structures represent an export mechanism by which
vesicles derived from the plastid can be moved around the
cell, either as part of a stromule network or as free vesicles.
Thus, the observations reported here are consistent with a
potential role for secretion from plastids via a stromuleassociated mechanism in glandular trichomes (Kim and
Mahlberg, 1997).
Although the true function of plastid stromules is unclear,
it appears likely that these structures may have several
different roles within the cell, dependent on the cell type.
One clear characteristic of stromule development is the
relative lack of stromules in chloroplast-containing cells,
and an increasing incidence of stromule production in cells
containing non-green plastids. We suggest that this correlation may, in part, relate to a plastid density-sensing
mechanism by which stromules are generated from plastids
in cells with relatively low plastid density to seek out
neighbouring plastids in order to sense overall population
size within that cell. Conversely, in chloroplast-containing
mesophyll cells, plastid density is high with all chloroplasts
touching several neighbours (Pyke, 1997). Consequently,
long-distance density-sensing stromules are not required.
Interestingly, red pigmented bodies of chromoplasts were
often observed in small clusters of two or three bodies from
which single stromules emanate. Thus, chromoplasts that
are immediately adjacent may not require stromules for
density sensing. Such a density-sensing mechanism in cells
could facilitate communication between plastids regarding
plastid division control and thus invoke division control at
the level of the plastid population rather than the individual
plastid (Pyke, 1997, 1999).
The present demonstration of the morphology of tomato
plastids and their associated stromules necessitates a reevaulation of the function of plastids in cells, particularly in
relation to the possibility of their co-ordinated interaction
via networked communication.
ACKNOWLEDGEMENTS
Thanks to Maureen Hanson (Cornell) for the gift of the GFP
clone, Syngenta Plant Science for providing tomato transformation facilities, Susan Anderson for assistance with
confocal microscopy, Rupert Fray for helpful discussions,
and Clair Baynton and Morag Kingshott for technical
support.
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