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Pavement cell chloroplast behaviour and interactions with other
organelles in Arabidopsis thaliana
Kiah A. Barton, Michael R. Wozny, Neeta Mathur, Erica-Ashley Jaipargas,
Jaideep Mathur 1*
1. Laboratory of Plant Development and Interactions, Department of
Molecular and Cellular Biology, University of Guelph, 50 Stone Rd., Guelph.
ON. N1G2W1. Canada.
* Author for correspondence
*JM
[email protected] Tel:1 (519) 824 4120 ext. 56636: Fax: 1
KB
[email protected]
MW
[email protected]
NM
[email protected]
EJ
[email protected]
Short description:
The spatiotemporal relationship between pavement cell chloroplasts and their
surroundings in Arabidopsis thaliana is presented.
JCS Advance Online Article. Posted on 20 March 2017
Journal of Cell Science • Advance article
(519) 837-1802
Abstract
Chloroplasts are a characteristic feature of green plants. Mesophyll cells
possess the majority of chloroplasts and it is widely believed that with the
exception of guard cells, the epidermal layer in most higher plants does not
contain chloroplasts. However, recent observations on Arabidopsis have
shown a population of chloroplasts in pavement cells that are smaller than
mesophyll chloroplasts and have a high stroma to grana ratio. Here using
stable transgenic lines expressing fluorescent proteins targeted to the plastid
stroma, plasma membrane, endoplasmic reticulum, tonoplast, nucleus,
mitochondria, peroxisomes, F-actin and microtubules we characterize the
spatiotemporal relationships between the pavement cell chloroplasts (PCC)
and their subcellular environment. Observations on the PCC suggest a
source-sink relationship between the epidermal and the mesophyll layers and
the Arabidopsis mutants glabra2 (gl2) and immutans (im) underscore their
developmental plasticity. Our findings lay down the foundation for further
investigations aimed at understanding the precise role and contributions of
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PCC in plant interactions with the environment.
Introduction
Plastids are multi-functional, pleomorphic organelles of purported
endo-symbiotic origin that display a characteristic double membrane envelope
in plants and green algae (Wise, 2007). Observations on the basic features of
plastids date back to the mid-seventeenth century (reviewed by Gunning et
al., 2007). All plastid types are believed to originate as colourless pro-plastids
in meristems (Schimper, 1883, 1885) and their subsequent differentiation
takes place according to tissue and developmental requirements (Jarvis and
López-Juez, 2013; Paila et al., 2015; Liebers et al., 2017). Early studies by
Schimper (1883, 1885) established the inter-convertibility of plastids from one
type to another. Presently, on the basis of pigmentation, chloroplasts are
distinguished from other plastids by the presence of chlorophyll, chromoplasts
by the predominance of other pigments, and leucoplasts by the absence of all
pigmentation.
In higher plants, the majority of chloroplasts are found in the leaf
mesophyll tissue and at the ultra-structural level display the classical thylakoid
membrane system of stacked thylakoid grana connected by intergranal
lamellae. However, chloroplasts in the bundle sheath cells of C4 plants are
often agranal (Bisalputra et al., 1969; Woo et al., 1970), suggesting that welldefined grana are not a requirement for identification as a chloroplast. Though
the majority of photosynthesis occurs in the mesophyll, the guard cells of most
plant species are recognized as having chloroplasts that possess an active
electron transport chain, are capable of fixing carbon dioxide and play an
2014).
In addition to guard cells the aerial epidermis consists of trichomes and
large pavement cells. It is widely believed that chloroplasts are absent from
the pavement cells of Arabidopsis and most other higher plants (Brunkard et
al., 2015; MacDonald, 2003; Smith, 2005; Bowes and Mauseth, 2008;
Solomon et al., 2010; Vaughan, 2013). However, the presence of chloroplasts
in pavement cells of Arabidopsis thaliana is well documented (Pyke and
Leech, 1994; Roberston et al., 1996; Vitha et al., 2001; Joo et al., 2005; Pyke,
2009) and was reinforced by recent observations (Barton et al., 2016).
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important role in stomatal opening and closing (Lawson, 2008; Lawson et al.,
Pavement cell chloroplasts (PCC) in Arabidopsis are comparable in
size to guard cell chloroplasts but are approximately half the size of mesophyll
chloroplasts (MCC). In the jigsaw shaped pavement cells in expanded
cotyledons and leaves of Arabidopsis, 9 to 15 PCC are found per cell, while a
single mesophyll cell may contain over 120 chloroplasts (Pyke and Leech,
1992, 1994; Barton et al., 2016). PCC are photosynthetically active and show
clear grana, but their chlorophyll autofluorescence signal is low compared to
MCC (Barton et al., 2016). A high stroma to thylakoid ratio in PCC makes
them easier to image than MCC using plastid-targeted fluorescent proteins, so
these plastids are sometimes used for visual studies of plastid responses
(Kwok and Hanson, 2004a; Higa et al., 2014; Caplan et al., 2015; Brunkard et
al., 2015). However, whether this small population shows responses that are
representative of all chloroplasts is uncertain and much remains unknown
about the spatiotemporal behaviour of PCC or their relationship with other
cellular components and compartments.
Here, using a range of fluorescent protein probes targeted to the
chloroplasts and other subcellular structures, we have investigated PCC and
their surroundings in Arabidopsis thaliana and contrasted PCC response to
light and sucrose with that of MCC. The creation of new transgenic plant
resources, and characterization of PCC from a cell biological, physiological
and developmental perspective lays down the foundation for further
investigations on the actual contribution of these small chloroplasts during
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plant interactions with the environment.
Results
Identifying the PCC and creating resources for their characterization
The presence of PCC in Arabidopsis was best appreciated when the
pavement cells were viewed from a lateral perspective. As shown in Fig. 1 this
view was easily achieved for pavement cells lying at the edges of cotyledons
and leaves. It allowed direct observation of the subcellular environment
around the PCC without interference from the fluorescence signal of
underlying mesophyll layers. The PCC are small when compared to the MCC,
however even from a lateral view, it can appear that some small chloroplasts
are in the mesophyll plane when the pavement and mesophyll cells are
closely interlocked. In order to confirm that the smaller chloroplasts were
located exclusively in the pavement cells, the Arabidopsis crooked (crk) and
wurm (wrm) mutants were used. In these mutants, epidermal cells in the
hypocotyl, cotyledons, and young leaves become abnormally elongated upon
detachment from each other along the long axis (Fig. 1A; Mathur, 2005).
Similar to wild type plants two chloroplast populations were visible in crk and
wrm, one consisting of small chloroplasts and the other of large chloroplasts.
The pavement cells that had curved out of the general epidermal plane in crk
and wrm plants exhibited small PCC clearly, while cells in the layers beneath
displayed only the larger MCC. (Fig. 1B; Movie S1). In wild type Arabidopsis
uncertainty about the exact location of chloroplasts in the epidermal or the
mesophyll layer can thus be resolved by observing their relative sizes.
A number of double transgenic and mutant Arabidopsis lines
different
targeted
fluorescent
probes
were
created
for
understanding PCC behaviour in relation to their subcellular environment
(Table 1). The following general observations were made on the interactions
between the highlighted organelles and the PCC.
Pavement cell chloroplasts and their surroundings
In 10 day-old cotyledons of soil grown tpFNR:GFP-RFP:ER plants, the
average cell depth for guard cells was estimated at 12.4 ± 0.6 μm, for
pavement cells at 38.3 ± 3.0 μm and for mesophyll cells at 53.8 ± 4.6 μm. In
comparison the pavement cells in petioles of the first leaves and cotyledons
were relatively elongated and thin with depths ranging from 12-17 µm.
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expressing
Observations in many plants taken at different times of the day on PCC
established that they were not always located in the same position with regard
to the upper cell boundary. While observations taken after an eight-hour dark
period showed up to 90% of the PCC located near the upper surface (Fig.
1C), they were localized mainly near the lower surface of the cell if the plants
had been exposed for a few hours to light intensity of about 120 µmol m-2s-1.
Due to their localization near the lower surface of the pavement cell these
chloroplasts appeared to lie on top of the MCC layer (Fig.1D). In both
locations the PCC extended and retracted stromules sporadically. These
observations on the relative sizes and positions of PCC and MCC were
reinforced using a line expressing a green fluorescent protein (GFP) targeted
to the plasma membrane (Fig. 2A).
Similar to the situation for the majority of organelles in plant cells
(Reviewed by Vick and Nebenführ, 2012; Geitmann and Nebenführ, 2015)
and in agreement with Kwok and Hanson (2004a), time-lapse imaging of the
double transgenic tpFNR:mEosFP-GFP:mTalin line showed PCC alignment
and movement along F-actin strands (Fig. 2B,C; Movie S2) and the F-actin
could be traced as a loose cage around the PCC (Fig. 2B). Compared to the
dynamic association between PCC and F-actin their spatiotemporal
relationship
with
microtubules,
investigated
using
a
tpFNR:mEosFP-
GFP:MAP4 line, was less clear. While a collapsed Z- stack of confocal images
conveyed the impression that the PCC were embedded between cortical
microtubules (Fig. 2D), in most optical sections the PCC were found in a
relationship with microtubules as stromules were often extended parallel to
the cortical microtubule array, but could also extend perpendicular to it (Fig.
2E). In the instances that alignment with neighbouring cortical microtubules
(Fig. 2E) was observed it was not sustained and did not occur along a
particular
microtubule.
Often
a
stromule
extended
across
multiple
microtubules and while retracting could pause at several locations.
In contrast to the microtubules, time-lapse observations in the
tpFNR:mEosFP-GFP:Vac line highlighted the tonoplast and revealed the PCC
were almost surrounded by, and could be tugged on by the movement of thin
vacuolar membrane tubules (Fig. 2F). For PCC located on the cell periphery
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distinct plane. PCC with extended stromules did not show a consistent
(Fig. 2G), the vacuole was closely appressed to the plastid on the side interior
to the cell.
In all cases, time-lapse imaging suggested that the vacuolar
membrane flowed loosely over the chloroplast surface (Fig. 2H; Movie S3).
Whereas a top down view (Fig. 2H) also suggested a very close association
between PCC and the vacuolar membranes, we were perplexed by the
presence of many areas that looked very similar in size to the PCC but did not
show the typical chloroplast fluorescence (Fig. 2I). We found the observations
with the vacuole probe reminiscent of earlier observations on chloroplasts
made using lines co-expressing tpFNR:GFP and an ER-targeted RFP
(Schattat et al., 2011). As reported earlier (Schattat et al., 2011) the PCC
were embedded within an ER cage surrounding the entire chloroplast (Fig.
2K) and both organelles moved in concert. Stromules extended sporadically
from the PCC and aligned closely with the ER mesh or extended along ERlined channels (Fig. 2L-arrowheads,M). In order to understand the PCCvacuole-ER relationship better we developed a double transgenic line coexpressing the vacuole targeted GFP and the ER targeted RFP. Using this
line it was found that the vacuole presses against and outlines other
organelles in a similar manner. Lateral views of PCC in the GFP:Vac-RFP:ER
line showed that the ER-cage was located between the vacuolar membrane
and the PCC (Fig. 2J) and that ER bodies could be amongst the organelles
appearing similar in size to PCC (Fig. 2J).
The vacuolar tubules also engulfed the nucleus and allowed us to
observe nucleus associated PCC (Fig. 2N). A line co-expressing the
15 ± 3 % (N=80 cells from 4 seedlings) of cotyledon pavement cells in 7 day
old seedlings, anywhere between 3 to 12 PCC could be found in the
perinuclear region (Fig. 2O). The nuclear association was often transient and
a variable number of PCC were observed reaching the perinuclear region or
moving away from it. In the remaining cells, the PCC were dispersed and
showed no clear nuclear association. Upon observing stromules extended by
the perinuclear PCC we were unable to find a clear indication whether the
stromules extended towards or away from the nucleus.
Each probe described here requires a more detailed assessment for
understanding the details of PCC response to different environmental stimuli.
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tpFNR:GFP and a nucleus-targeted RFP was developed and showed that in
However, the PCC are clearly present in a narrow cytoplasmic sleeve
appressed to the pavement cell boundary, comprising of the plasma
membrane and the subtending cytoskeleton, by the dynamic vacuole. The
narrow cytoplasmic sleeve with PCC is also populated by small organelles like
mitochondria and peroxisomes and PCC relation with these organelles was
investigated next.
Assessing PCC interactivity with mitochondria and peroxisomes
Published biochemical (reviewed by Raghavendra and Padmasree,
2003; Hodges et al., 2016) as well as microscopy-based findings (Frederick
and Newcomb, 1969; Sage and Sage, 2009; Gao et al., 2016; Jaipargas et
al., 2016) suggest close interactivity between chloroplasts, peroxisomes and
mitochondria. Stromules extended from plastids have been specifically
implicated in such interactions (Kwok and Hanson, 2004a) and a relation
between stromule extension and the cell size has been proposed (Waters et
al., 2004). Between 9 and 15 small chloroplasts are found in a pavement cell
compared to up to 120 chloroplasts of nearly twice their size in mesophyll
cells (Pyke and Leech, 1992, 1994), thus the pavement cells provide a much
larger total cell volume to total chloroplast volume ratio as compared to the
mesophyll cell. Based on published literature (Waters et al., 2004) there
seemed to be a high likelihood of observing stromules and their interactions
with small organelles in these cells. We created a double transgenic line
tpFNR:YFP-mitoGFP (Logan and Leaver, 2000) to investigate the interactivity
line (Fig. 3B) for observing PCC interactions with peroxisomes.
Both mitochondria and peroxisomes appear to come into close
proximity with the PCC. Time-lapse image series showed that associations
with PCC could range from fleeting single-frame encounters to sustained
encounters of over 10 seconds. However, small organelles that were
permanently associated with PCC were not observed.
Mitochondria and
peroxisomes can both show punctate or elongated morphologies. Elongated
organelles were very dynamic and the region in contact with the plastid could
change over time. Fig. 3C summarizes the variations in organelle shape and
position that were seen. In order to determine if these organelles associated
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between mitochondria and PCC (Fig. 3A) and a tpFNR:mEosFP-GFP-PTS1
more frequently with stromules or with the plastid body, the total number of
mitochondria or peroxisomes that came into close apposition with PCC
producing stromules over time was counted and averaged. The average
number of mitochondria or peroxisomes in close juxtaposition to the PCC
main body or to the extended stromule did not differ (Fig. 3D). In the PCC
considered in these time-lapse image sets, the average perimeter that was
available for association with other organelles did not differ between the
plastid bodies and the stromules (Fig. S1). Having observed the general
subcellular environs of the PCC we focused next on the implications of their
position relative to the MCC.
PCC positioning over the MCC creates a physiological relationship
between the two layers
PCC have photosynthetic capability, however the number and
thickness of grana and the chlorophyll content in PCC is low compared to
MCC (Barton et al., 2016). In Arabidopsis the general stromule formation
frequency of PCC is known to increase during the day (Schattat et al., 2012a;
Brunkard et al., 2015) but whether this is representative of the response of
MCC has yet to be studied. Observations taken separately on the two cell
layers in 6 week old short day grown plants showed that the stromule
frequency of PCC increased in response to light exposure and continued to
rise through the 8 hour light cycle. By contrast the MCC had a consistently low
stromule formation frequency that showed no apparent change throughout the
Exogenous sugar feeding is known to increase stromule formation
frequency (Schattat and Klösgen, 2011), and was used to determine if PCC
and MCC stromule formation responses differed under other conditions. Intact
21 day old Arabidopsis plants that were floated in 40mM sucrose in the dark
showed an increase in stromule frequency of both PCC and MCC (Fig. 4B).
Control treatments indicated that neither the immersion in water nor the
prolonged darkness influenced the basal stromule levels. PCC stromule
frequency was consistently higher than that of MCC, but for both populations
the increase began within the first hour, and continued to rise throughout the 5
hours of treatment (Fig. 4B). However, the stromule formation response from
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day (Fig. 4A).
MCC was almost entirely inhibited when the plants were exposed to light
during the sucrose treatment, while the PCC response was unchanged from
the dark treatment (Fig. 4C, D).
During our light and sugar experiments, chlorophyll autofluorescence
confirmed that the plastids of all pavement cells observed were indeed
chloroplasts. However, reports of leucoplasts in pavement cells of Arabidopsis
do exist, so we further investigated under what conditions this could occur and
how the behaviour and appearance of epidermal leucoplasts differ from PCC.
A
developmental
perspective:
Exploring
PCC
and
plastid-
interconvertibility
Leucoplasts, by definition (Schimper, 1883; 1885), do not contain
chlorophyll and are stroma-rich. When using the stroma-targeted tpFNR-GFP
probe (Marques et al., 2004; Schattat et al., 2011), leucoplasts appeared
uniformly green fluorescent (emission 510-520 nm), while in chloroplasts the
green stromal fluorescence surrounded the chlorophyll autofluorescence of
the grana (emission 650-750nm). This allowed us to monitor the presence of
both plastid types in pavement cells during development. We first investigated
whether chloroplasts are the default plastid type in the epidermis of
Arabidopsis.
Observations on Arabidopsis leaves in the tpFNR:GFP line revealed
that unlike in the chloroplast-containing guard and pavement cells, trichome
plastids are leucoplasts (Fig 5A). As trichomes are initiated from protodermal
2005), if the chloroplast represents the standard plastid type in epidermal
cells, the chlorophyll must be lost from trichome plastids as the cell
differentiates. In the glabra2 (gl2) mutant of Arabidopsis the trichome
differentiation is initiated, but the majority of trichomes do not remain
committed to the trichome fate and either mature into small spikes (Fig. 5B,C)
or collapse into large misshapen cells very similar to pavement cells (Fig. 5D;
Rerie et al., 1994). In a gl2 GFP:mTalin line where the green fluorescence
provides cellular context to the chlorophyll auto-fluorescence, the aberrant
trichomes exhibit chloroplasts rather than leucoplasts (Fig 5E,F). Therefore,
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cells in expanding leaves (Szymanski et al., 2000; Schellmann and Hülskamp,
guard cells, pavement cells and trichome cells that have not completely
differentiated all contain chloroplasts in Arabidopsis.
We next looked for conditions under which epidermal leucoplasts might
be observed. Leucoplasts were not seen in cotyledon pavement cells of
healthy 7 to 12 day old tpFNR:GFP plants grown either in the soil or on agarsolidified medium, nor were they seen in green leaves of older plants.
Pavement cell leucoplasts were only found in regions of these plants with
mechanical damage or in plants that showed high anthocyanin accumulation
indicative of stress. Chloroplasts remained the predominant population in
older cotyledons between 14 and 28 days.
Seldom, uniformly green
fluorescent leucoplasts and senescing chloroplasts with a distinct separation
of GFP and chlorophyll fluorescence but an intact boundary could be seen
(Fig. 6A). In chlorotic tissue of 6 week old plants, the chlorophyll signal was
minimal, but still present in most epidermal plastids. Similar to senescing
cotyledons, leucoplasts could be seen (Fig. 6B), but were extremely
infrequent. It is therefore possible to see pavement cell leucoplasts under
some conditions, but the occurrence is rare. Since we were unable to
consistently observe leucoplasts in pavement cells in young wild type tissue,
we used the immutans (im) mutant of Arabidopsis (Rédei, 1967; Wetzel et al.,
1994) for a thorough comparison of PCC and leucoplast appearance and
behaviour.
The im mutant is variegated with bright green chloroplast-containing
regions randomly interspersed with white sectors that contain leucoplasts as
1994). This mutant thus provided an ideal tool to investigate the different
appearances of leucoplasts and chloroplasts within the same tissue.
Introduction of the tpFNR:GFP probe into im allowed easy identification of
leucoplasts by their complete lack of chlorophyll autofluorescence (Fig. 6E).
The shape of the various plastid types was clearly different, with leucoplasts
displaying an elongate and irregular form as compared to the consistent,
round to oval shape of chloroplasts (e.g. Fig. 1C). PCC in green sectors, apart
from the occasional extension of stromules, maintained their lens-like shape
over time, whereas leucoplasts exhibited a dynamic morphology. In rare
cases, individual leucoplasts did maintain a round morphology over time.
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well as intermediate plastid forms (Fig. 6C,D; Rédei, 1967; Wetzel et al.,
Observations on the im leaf epidermis provided another interesting insight
(Fig. 6F) on plastids. No chlorophyll signal was visible in any epidermal cell
type in the white sectors and even within a pavement or guard cell, the
leucoplast size varied considerably. Similarly, chloroplasts of different sizes
were visible in the pavement cells of green sectors. While the presence of
chlorophyll in green sectors reinforced our earlier observations on wild type
plants, it became apparent from the im mutant that plastid size cannot be
considered as a consistent criterion for identification of either leucoplasts or
chloroplasts within a tissue.
Based on our investigations on the gl2 and im mutants it is clear that there
is
tremendous
condition-dependent
inter-convertibility
between
the
chloroplasts and leucoplasts. In wild type Arabidopsis, whereas pavement
cells in young cotyledons and leaves possess small chloroplasts, cells of older
tissues may have an increased number of leucoplasts.
Discussion
The presence of chloroplasts in epidermal pavement cells of
Arabidopsis has already been established (Pyke, 2009; Barton et al., 2016).
The present work aimed to describe fresh resources and the basic subcellular
relationships for PCC to facilitate further study on this under-researched
subpopulation of chloroplasts.
Based on the probes that have been used, a general picture of the
PCC in Arabidopsis has emerged. They are found in a thin cytoplasmic sleeve
and a reinforcing cytoskeletal mesh. PCC and stromules exhibit clear
alignments with actin filaments. Interactions with the actin cytoskeleton have
been suggested to play a role in both stromule extension and whole plastid
movement (Kwok and Hanson, 2004a; Kadota et al., 2009). Indeed, the
coordinated movement of the perinuclear plastids by the actin cytoskeleton
has been reported to play a role in nuclear movement during the blue-light
avoidance response (Higa et al., 2014). Despite this, we saw clustering of
PCC around nuclei in only a small proportion of pavement cells, suggesting
that nuclear movement either does not require PCC presence or requires a
mass migration of PCC to the nucleus before it can occur. The actin
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delimited on its exterior by the cell boundary consisting of plasma membrane
cytoskeleton also supports the cortical ER and the vacuolar membrane
(Higaki et al., 2006; Ueda et al., 2010). The vacuolar membrane appears to
press cortical PCC against the cell periphery and almost completely encloses
more cell-central PCC. Though we saw that rapid vacuolar movement was
often accompanied by the appearance of ‘tugging’ on PCC, the vacuole
appears primarily to move freely over the plastid surface.
This is in
accordance with the idea of a shared association with the actin cytoskeleton
but does not suggest a role for the vacuole in directed PCC or stromule
movement. In contrast, as reported by Schattat et al. (2011) and reconfirmed
here, the PCC and extended stromules are firmly enmeshed in the cortical ER
and plastid behaviour appears to directly correlate with ER re-arrangement.
Unlike the previous organelles discussed, no clear spatial association of the
microtubules with PCC could be seen. Microtubules form a tight array of
largely parallel filaments that traverse the cell periphery (Reviewed in Dixit
and Cyr, 2004), and PCC seemed to sit below the plane of this array, with
stromules extending either parallel or perpendicular to it. The potential for a
microtubule effect on PCC should however be investigated further, as the ER
has been implicated in stromule extension (Schattat et al., 2011) and an effect
of microtubules on ER rearrangement has been reported (Hamada et al.,
2014).
The relationship between the outer membranes of the PCC, the ER
and the vacuole creates a very dynamic field for membrane contacts and
exchange of metabolites and ions. Ongoing investigations on the membrane
contacts between the PCC and the surrounding endomembranes facilitated
that will be reported elsewhere.
All plastids are known to produce stromules (reviewed by Gray et al,
2001; Hanson and Sattarzadeh, 2011; Schattat et al., 2015) and these
extensions have been suggested as interaction platforms for organelles such
as nuclei, mitochondria and peroxisomes (Kwok and Hanson, 2004a, 2004b).
Our present observations strongly suggest that these organelles associate
only occasionally and rather transiently, and that mitochondria and
peroxisomes show no preference for association with the stromule over the
plastid body. On this basis we do not consider that there is a specific
subpopulation of mitochondria and peroxisomes that are targeted particularly
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by the double transgenic lines reported here are providing interesting insights
to the PCC body or stromule and maintain a sustained presence around them.
However, though the individual perimeters of the stromule and plastid body
were comparable, extension of a stromule does increase the total plastid
perimeter. Though no preference is shown for association with a stromule, its
extension may increase the total number of organelles with which a plastid
can interact. Accumulation of plastids has been observed in the perinuclear
region of hypocotyl cells and dark-adapted leaves of Arabidopsis (Kwok and
Hanson, 2004b; Higa et al., 2014), and in pathogen challenged cells of
tobacco (Caplan et al., 2008; Krenz et al., 2012; Caplan et al., 2015), however
our present observations do not suggest the presence of a sustained PCC
population around the nuclei under standard growth conditions. In addition,
we have been unable to support the idea that stromules extended by the PCC
in the perinuclear region display a specific configuration in relation to the
nucleus. Whether the perinuclear aggregation of chloroplasts occurs in
mesophyll cells in Arabidopsis, as well as whether this phenomenon is
specific to stressed plants or plays a role in normal growth and development
remain to be investigated.
A major insight provided from this work involves the inter-relationship
between chloroplasts and the differences in plastid behaviour between the
pavement and mesophyll cells. In a model that considered PCC to be
leucoplasts it was proposed that the epidermal layer acts as a sink for sugars
produced by the mesophyll (Brunkard et al., 2015). In principle all chloroplasts
are capable of photosynthesis, but their output of photosynthates might differ
size and number (Pyke and Leech, 1994; Barton et al., 2016), it is likely small
compared to that of the underlying MCC. Therefore, recognizing the plastids
in pavement cells as chloroplasts does not negate a potential source-sink
relationship between these two tissues. In order for continuous export of
photosynthates from MCC and efficient photosynthesis, mesophyll cells must
shuttle sugars to sink tissues (Ainsworth and Bush, 2011). The pavement
cells, being close in proximity and potentially low in sugar production, could
provide a readily accessible sink. Experiments done by Schattat and Klösgen
(2011) showed that sugar induces stromule formation, so an accumulation of
sugar in sink tissues may be accompanied by an increased stromule
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considerably. The sugar output from PCC is unknown but given their small
frequency. In observations throughout the photosynthetic period, we found
that PCC stromules increased in number throughout the day but that MCC
maintained a consistently low stromule frequency. This observation, alongside
the sugar responsiveness of stromules, suggests that while MCC mainly
exported their photosynthates, the PCC responded to the consequent
increase in levels of cytosolic sugar within the pavement cells. The idea was
further tested by inducing stromules with exogenously fed 40 mM sucrose
(Schattat and Klösgen, 2011; Schattat et al., 2011b). The stromule formation
frequency increased significantly in PCC regardless of whether light was
given during treatment. Interestingly, an increase in MCC stromule frequency
occurred in the dark, but not when plants were exposed to light during
treatment. This supports a model where mesophyll chloroplasts are capable
of sugar-induced stromule induction, but while photosynthesis is active
continuous export of sugars from the cytosol prevents sufficient accumulation
for induction. We therefore propose that pavement cells act as a sink for
sugars from the mesophyll during the daylight period. Investigations are
underway to explore the physiological sugar regulated inter-chloroplastic
relationship further. Another important implication of these results is that PCC
behaviour is not representative of the majority of leaf chloroplasts under all
conditions. Therefore, care should be taken when choosing an experimental
tissue, and before applying a model based on PCC to all chloroplasts, the
individual PCC and MCC responses should be compared.
Our results also highlight the potential effect of growth and treatment
Previous studies in Arabidopsis and
Tobacco on stromule responses throughout the day-night cycle showed an
increase in stromule frequency within the first few hours of light exposure and
then consistent stromule frequency throughout the day (Schattat et al., 2012;
Brunkard et al., 2015). This is in contrast to our observations of a gradual
increase in stromules over the course of the entire day. Interestingly, both
previous studies grew their plants under a 12 h light / 12 h dark photoperiod,
while we used an 8 h light / 16 h dark. If the diurnal changes in cellular sugar
status were responsible for the stromule response then photoperiod would
certainly have an effect, as plant sugar metabolism adjusts to compensate for
a longer night (Suplice et al., 2014). Photoperiod, as well as the time of day
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conditions on stromule responses.
that tissue is collected, should therefore be taken into consideration when
comparing observations of stromule frequency between experiments.
Similarly, our observations of increased stromules in the mesophyll in
response to sugar treatment appear to conflict with the statement by Brunkard
et al. (2015) that mesophyll chloroplasts are not sugar responsive. However,
as demonstrated here, mesophyll stromules are only induced by sucrose
treatment in the dark. Brunkard et al. (2015) do not specify the lighting
conditions for their treatment, but if they carried out their treatments in the
light, then both observations are in accordance. It is therefore apparent that
many factors, including photoperiod, time of observation and light exposure
during treatment, influence plastid behaviour and that thorough reporting of all
conditions is necessary to allow for comparison between studies.
The suggested link between PCC behaviour, photosynthesis and sugar
regulation also led us to explore the developmental aspects of chloroplast
formation. The present view traces all plastids to a population of pro-plastids,
which are colourless and so by definition, constitute leucoplasts. Further
development
of
these
colourless
plastids
into
chlorophyll-containing
chloroplasts occurs in a tissue and cell specific manner and requires input
from nuclear encoded proteins (Jarvis and Lopez-Juez, 2013; Liebers et al.,
2017). The linear route of leucoplasts and etioplasts greening into
chloroplasts is thus well supported. Studies on plastids in the L1 layer of the
shoot apical meristem and the epidermis of developing embryos suggest that
this conversion occurs early in the epidermal cells, as either stacked grana or
et al., 2010; Charuvi et al., 2012).
Our observations with the gl2 mutant
support chloroplasts as the standard plastid type in epidermal cells, as the
presence of chloroplasts in the under-developed trichomes suggests the
presence of PCC in the protodermal cells from which trichomes originate. It
follows that loss of chlorophyll is responsible for the presence of leucoplasts in
wild type trichomes, and that chloroplast to leucoplast conversion is part of
normal development.
We found that this phenomenon also occurs in
senescent cells and tissues and in these cases is dependent upon a change
in the cell physiology. As senescence is accompanied by breakdown of
chlorophyll (Hörtensteiner, 2006) and the chlorophyll signal is relatively low in
Journal of Cell Science • Advance article
chlorophyll autofluorescence were seen in the plastids of these tissues (Tejos
PCC (Barton et al., 2016), they are likely to be among the first plastids in
which chlorophyll loss is visible during senescence. Based on our
observations, it is clear that investigations of PCC in Arabidopsis must always
take the growing conditions, tissue health and developmental stage of the
plant into consideration.
The use of the im mutant (Rédei, 1967; Wetzel et al., 1994) allowed us
to reliably observe leucoplasts in pavement cells and indicated another facet
of the leucoplast-chloroplast relationship. The two plastid types show
considerable differences in behaviour but we determined that neither shape
nor size was sufficient on its own to distinguish between chloroplasts and
leucoplasts. This mutant also provides an interesting tool for future research
to test, in a horizontal context, our hypothesis that differences in stromule
response can be indicative of a source-sink relationship between cells. The
white sectors with leucoplasts and the green sectors with photosynthetic
chloroplasts lie side by side on a leaf in this mutant. Since the white sectors
are non-photosynthetic, they represent sink tissues that should show a
stromule response to accumulation of photosynthates from juxtaposed green
regions.
Conclusions
Here we have provided a cell biological characterization of the small
sub-population of chloroplasts in epidermal pavement cells of Arabidopsis.
Plastid responses to changes in the cellular milieu and the phenomenon of
basic characterization of chloroplasts in the pavement cells of Arabidopsis
triggers several interesting questions and new approaches to understand their
functional significance and their relationship with chloroplast populations in
contiguous and subtending cells.
Journal of Cell Science • Advance article
plastid inter-convertibility have been highlighted through our observations. Our
Methods
Plant material and growth conditions
Single
and
double
transgenic
lines
were
created
through
Agrobacterium mediated floral dip transformation (Clough and Bent, 1998) or
through crossing. A complete list of plant lines used is provided in Table 1.
The four different conditions for plant growth were as follows: (1) growth in
sealed tissue culture boxes for 7-12 days or 21 days on sterilized Sunshine
mix LA4 under a long day light regime (16 h light / 8h dark) for experiments on
stromule frequency in response to sucrose; (2) growth for 7-12 days in petri
dishes containing Murashige and Skoog basal medium (1962; Sigma M404)
with B5 vitamins and 3% sucrose under a long day light regime for
observations of the interactions between PCC and other organelles; (3)
growth in pots for 6 to 7 weeks on Sunshine mix LA4 in a controlled chamber
under a short day light regime (8 h light/16 h dark) for observations on diurnal
plastid responses and leucoplast conversion in leaves. Diurnal stromule
observations were taken on healthy leaves, while senescence observations
were taken on older chlorotic leaves. All plants were grown under 120 μmol
m-2s-1 light and at a temperature of 21°C.
Confocal microscopy
All fluorescence imaging was done on whole plants, with the exception
of imaging of leaves over 3 weeks of age, which were detached and imaged
TCS-SP5 confocal microscopy system equipped with a 488 nm Ar laser and a
543 nm HeNe Laser. GFP fluorescence was acquired between 500-520 nm,
RFP fluorescence between 570-620 nm, YFP fluorescence between 530-550
nm and chlorophyll autofluorescence between 660-750 nm. mEosFP was
photo-converted using a mercury lamp and the Leica fluorescence filter set D
(Excitation 355 to 425 nm).
The entire field of view was exposed to the
conversion light for 10 to 15 seconds. All confocal images were acquired at a
resolution of 1024 x 512 or 1024 x 1024 pixels. Three-dimensional (X,Y,Z)
stacks were collected with a step-size of 0.99 μm and successive frames in
X,Y,T time lapse series were 1.935 seconds apart.
Journal of Cell Science • Advance article
immediately. The plants were mounted in water and imaged using a Leica
Images were processed using proprietary Leica (LSM SPII) software,
ImageJ (Schneider et al., 2012) and Adobe Photoshop CS6. Quick time 7 and
Videator v2 (Stone Design Corp., New Mexico) were used for assembling and
annotating time-lapse movies.
Scanning electron microscopy (SEM)
SEM was carried out on unfixed, uncoated samples taken directly from
the green house and observed using a Hitachi Tabletop TM-1000 system
(Hitachi High-Technologies Corp., Tokyo).
Calculation of cell depths
Cell depths were calculated in the FNR-GFP RFP-ER background to
allow visualization of the plastid populations and the cortical ER surrounding
the cell borders. Z-stacks were taken with a step size of 0.4 μm from the
upper epidermal surface to the bottom of the mesophyll cells in the cotyledons
of 8 individual 10 day old seedlings.
In ImageJ (Schneider et al. 2012),
orthogonal views allowed the calculation of cell depth of between 15 and 20
cells of each type (pavement, guard or mesophyll) per stack based on the
number of steps required to traverse the cell.
Analysis of PCC and small organelles
Time-lapsed images were collected for 41 to 321 frames. 1-3 PCCs per image
set had stromules that remained in focus for the frames analyzed. Collection
were followed for analyzing mitochondria and peroxisome encounters with
PCCs respectively. The number of mitochondria or peroxisomes closely
appressed to the PCC body or stromule was counted manually for each frame
of the image sets using the ImageJ Cell Counter plugin (Kurt De Vos, 2001).
The total number of close encounters between PCCs and either mitochondria
or peroxisomes was summed and averaged over the total number of frames
analyzed. 95% confidence intervals (CI) for the estimated means were
calculated for the data. The perimeter of the body and stromule of PCCs was
measured in the first frame of the time-lapse image sets used for counting the
Journal of Cell Science • Advance article
time for each frame was 1.935 seconds. A total of N=38 and N=10 PCCs
average number of mitochondria juxtaposed to PCCs and the mean
perimeters were reported with 95% confidence intervals.
Stromule frequency: Treatment, image acquisition, calculation and
statistical analysis
Treatment of seedlings with 40mM sucrose or water controls was
performed by carefully excising the entire plant from the soil and floating the
plants in solution for 3 hours in either a dark chamber or under normal growth
lighting. For consistency, plants were always taken at the end of the night
cycle to begin treatment. For each condition under which stromule frequency
was measured, N= 16 z-stacks were taken with a step size of 0.99 μm
through the epidermis and the upper region of the mesophyll on the adaxial
surface of the most expanded leaves. Standard deviation z-projections were
obtained using ImageJ (Schneider et al., 2012) and the number of plastids
exhibiting or lacking stromules was counted manually for both the PCC and
MCC populations in each image. The calculated stromule frequencies were
transformed with the arcsin(√Freq) function according to Schattat and
Klösgen, (2011). 95% confidence intervals (CI) for the estimated mean and
statistical significance (Two-way ANOVA with post-hoc Bonferroni test) were
assessed using the transformed data.
The mean and CI were back-
transformed for representation in the figures.
Acknowledgements
for Mito:GFP, Sean Cutler for the GFP:Vac line Q5, Steven Rodermel for the
im mutant, Martin Schattat for tpFNR:YFP and Rob Mullen for the RFP-NLS
DNA probe. JM and KB gratefully acknowledge funding by NSERC, Canada.
Journal of Cell Science • Advance article
We thank Peter Dörmann for seeds of 35S-NPC4:GFP, David Logan
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Figures
Figure 1. The presence of pavement cell chloroplasts in Arabidopsis
A. Scanning electron micrograph of the petiole region of a cotyledon in the
wrm mutant where epidermal cell detachment along the long axis results in
B. A confocal image shows plastids with chlorophyll fluorescence (false
coloured magenta) in a detached epidermal cell (arrow) from a cotyledon
petiole in the wrm mutant. Chloroplasts in the mesophyll layer (arrowhead)
are larger compared to the PCC.
C. Representative image from a cotyledon petiole taken soon after a 8h dark
period shows chloroplasts in the top portion of the pavement cell.
Journal of Cell Science • Advance article
single epidermal cells (arrow) curling out.
D. An image from a cotyledon petiole taken after 4h of exposure to light
shows PCC aligned along the bottom of the epidermal cell and thus appearing
to lie over the MCC.
Journal of Cell Science • Advance article
Size bars - B, C = 25; D = 50; A = 100µm.
Figure 2. A general fluorescent protein aided assessment of pavement
A.
A collapsed 3D-confocal image stack of a leaf from a transgenic line
expressing a plasma membrane targeted GFP (35S-NPC4:GFP) shows the
relative sizes and positions of PCC (arrows), chloroplasts in guard cells and
the mesophyll chloroplasts (e.g. square box).
B. A representative image from a leaf showing the general F-actin
cytoskeleton around PCC (arrow) and an inset ‘b’ showing a magnified view of
F-actin cage around a single PCC. The underlying blue layer contains the
high chlorophyll-containing MCC.
Journal of Cell Science • Advance article
cell chloroplasts and their surroundings
C. Image from the double transgenic tpFNR:mEos-GFP:mTalin line shows the
main chloroplast body and the elongated stromule aligned with a F-actin
strand.
D. Most PCC do not appear in the same section as cortical microtubules and
a collapsed 3D stack of confocal images suggests their random dispersal in
the cell at different planes. Relative to the PCC the MCC (arrow) appear large.
E. A collapsed stack of 3D confocal images suggests that stromules (St) may
align transiently with CMt over short distances.
F.
PCC (chlorophyll fluorescence in blue) appressed against the upper
boundary of a pavement cell by the underlying green fluorescent vacuolar
tubules and the underlying MCC.
G. Lateral view of a pavement cell in the tpFNR:mEosFP-GFP:Vac line
showing the peripheral localization of PCC (arrowheads) with extended
stromules and subtending vacuolar tubules.
H. A top down view of a pavement cell shows PCC with stromules engulfed by
vacuolar tubules (See also Movie S3)
surrounding achlorophyllous structures of the same size as PCC.
J. Image acquired on an RFP:ER-GFP:Vac double transgenic shows the
profile of an achlorophyllous structure suggested in ‘I’ is due to the vacuole
surrounding an ER-body.
K. Image of a PCC with an ER cage similar to that observed around MCC.
L. Image of a chloroplast-containing PCC-body (blue) enmeshed in an ER
cage (red) and with a contiguous ER channel (arrowheads).
Journal of Cell Science • Advance article
I. An image from the GFP:Vac transgenic line shows the membrane
M. Image of a PCC (chlorophyll – blue) from the tpFNR:GFP-RFP:ER line with
green fluorescent stromules extended into the ER channels.
N. Image of the vacuolar tubules engulfing the nucleus and three PCC
(arrowheads) in the peri-nuclear region.
O. Image of PCC with and without extended stromules clustered around a
nucleus in a tpFNR:GFP-RFP:NLS line.
Abbreviations: CMt - cortical microtubules; ER - endoplasmic reticulum; Factin - filamentous actin; GC - Guard cells; PCC – pavement cell chloroplasts;
MCC- mesophyll cell chloroplasts; Nu – nucleus; St - Stromule; Vt - Vacuolar
tubule;
Journal of Cell Science • Advance article
Size bars (µm): A = 25; B – H, I, J, N, O = 10; b, K, L, M = 5.
Figure
3.
Observations
on
PCC
in
tpFNR:YFP-mito:GFP
and
tpFNR:mEosFP-GFP-PTS1 double transgenic lines of Arabidopsis.
A. A snapshot from a time-lapse image sequence showing PCC (yellow) with
stromules (St) and mitochondria (green) to estimate their possible interactivity.
B.
A snapshot of PCC (magenta) with stromules (red) and peroxisomes
C. A diagrammatic depiction of the locations of the two organelles (red depicts
either mitochondria or peroxisome) on either the main plastid body or the
extended stromule that were used to build data for insights on their
localization patterns.
D. The average number of mitochondria (Mt) or peroxisomes (Px) in close
juxtaposition to a PCC body or an extended stromule. Error bars represent
the 95% CI for the estimated mean (N=38, N=10 respectively)
Journal of Cell Science • Advance article
(green) co-visualized for estimating their possible interactivity.
Letters indicate significant differences between groups (P < 0.05) within a light
treatments or cell type (a to d for PCC or light treatment, r to u for MCC or
dark treatment).
An asterisk indicates a significant difference (P < 0.05)
Journal of Cell Science • Advance article
between light treatments or cell types.
Figure 4. Using stromule formation to assess the physiological
relationship between epidermal and mesophyll layers
plants grown under short day conditions. Error bars represent the 95% CI for
the estimated mean (N=16).
B. Hourly change in stromule frequency of PCC and MCC in response to
treatment with a 40 mM sucrose solution. Error bars represent the 95% CI for
the estimated mean (N=16)
C. D. Stromule frequency of PCC (C) and MCC (D) in 21 day old plants
treated with 40 mM sucrose or a water control for 3 hours. Plants were either
Journal of Cell Science • Advance article
A. Stromule frequency of PCC and MCC over an 8h light period in 6 week old
kept in the dark or exposed to 120µmol m-2s-1 light during the treatment. Error
bars represent the 95% CI for the estimated mean (N=16).
Letters indicate statistically significant groups (p < 0.05) within cell types or
treatments (a-d for PCC or Light, r-u for MCC or Dark). Asterisks indicate a
Journal of Cell Science • Advance article
difference (p < 0.05) between cell types or treatments.
trichome cells in Arabidopsis expressing tpFNR:GFP and in the glabra2
(gl2) mutant epidermal cells.
A. A flattened z-stack of 10 confocal images shows the base of a trichome,
the epidermis and the mesophyll layers in an Arabidopsis (Columbia ecotype)
plant expressing tpFNR:GFP. Panels 1, 2, 3, 4 show GFP (green) and
chlorophyll autofluorescence (red), a bright field image and a merged image,
respectively. Note the presence of chlorophyll fluorescence in the pavement
cells of the epidermal layer but its complete absence in the trichome cell
Journal of Cell Science • Advance article
Figure 5. Images showing leucoplasts and chloroplasts in and around
(panel 2). As shown by the stroma-targeted tpFNR:GFP probe mature
trichomes
in
Arabidopsis
possess
leucoplasts
only
(panel1;
green;
arrowhead).
B. A scanning electron microscope image of a stubby trichome ( * ) that has
not completed its differentiation in the gl2 mutant.
C. A short, collapsed trichome in gl2 similar to ‘B’ has plastids that exhibit
chlorophyll autofluorescence (red) comparable to the fluorescence of
chloroplasts in the sub-epidermal layers.
D. A scanning electron microscope picture of a cell on a gl2 leaf ( * ) that
appears to have entered the trichome differentiation pathway but did not
progress to branching and maturation stages. Its collapse and enlargement
suggest its continuation / reversion into a pavement cell fate.
E. Image of a gl2 trichome similar to ‘D’ that has not completed its
differentiation shows plastids with a chlorophyll signal (red) alongside a
GFP:mTalin F-actin fluorescence (green).
F. A gl2 mutant line expressing a plasma-membrane targeted GFP probe
shows chlorophyll-containing plastids (red) in a putatively aborted trichome.
Journal of Cell Science • Advance article
Size bars: A, B = 50 µm; C - F = 25 µm.
clear differentiation between leucoplast and chloroplasts.
Journal of Cell Science • Advance article
Figure. 6. Images from Arabidopsis wild type plants and mutants allow
A. A group of physiologically compromised swollen PCC in a senescent
cotyledon exhibit the clear separation of their GFP highlighted stroma and the
chlorophyll contents.
B. An image from a pavement cell in a senescing 16 day old cotyledon of an
Arabidopsis plant expressing tpFNR:GFP shows several green leucoplasts
(e.g. arrowhead) alongside a plastid exhibiting a green-red mix (panel 4)
suggesting the presence of chlorophyll (red; panel 2).
C. Image of young plants of the Arabidopsis variegated mutant im showing
the characteristic green (g) and white (w) sectors containing chloroplasts and
leucoplasts, respectively.
D. Image of a leaf of the im mutant transformed with stroma targeted
tpFNR:GFP shows green fluorescent sectors that contain mostly leucoplasts
and regions with chloroplasts whose fluorescence is false colouration.
E. A compressed confocal image stack of a portion of a leaf petiole in im
shows lens-shaped chloroplasts (false coloured blue) and variously shaped
and elongated green fluorescent leucoplasts that clearly lack chlorophyll.
F. Confocal image of a variegated leaf from im shows chlorophyll
in cells on the upper left that represent a white leucoplast rich sector whereas
the right side of the image shows mesophyll chloroplasts. Chloroplasts that
are intermediate in size between guard cell chloroplasts (boxed areas) and
mesophyll chloroplasts are visible in the pavement cells in the lower left leaf
sector. Chloroplasts in guard cells (similar to the 3 in boxed areas) appear in a
typical curved pattern as compared to the scattered chloroplasts in
neighbouring pavement cells.
Size bars: A, B=5 µm; C=1 mm; D=100 µm; E= 20 µm; F =25 µm.
Journal of Cell Science • Advance article
fluorescence (blue) against a bright field background. Chloroplasts are absent
Table 1. Transgenic lines and mutants of Arabidopsis used for assessing the
subcellular environment around pavement cell chloroplasts
Line
Single transgenics
Highlighted subcellular compartment
Reference
tpFNR:GFP
Plastid stroma green
tpFNR:YFP
Plastid stroma yellow; Replaced GFP by
YFP
Plastid stroma green to red photoconvertible
Plasma membrane green
Marques et
2003, 2004
Schattat et
2011a
Schattat et
2012b
Gaude et
2008
tpFNR:mEosFP
35S-NPC4:GFP
al.,
al.,
al.,
al.,
Double transgenics
tpFNR:GFP x RFP:ER
Plastid stroma green, ER lumen red
tpFNR:GFP x RFP:NLS
Plastid stroma green, Nuclear interior red
tpFNR:YFP-T-Mito:GFP
Plastid stroma
matrix green
tpFNR:mEosFP x GFP:PTS1
Plastid
stroma
green
to
red
photoconvertible,
Peroxisome
matrix
green
Plastid
stroma
green
to
red
photoconvertible, Vacuolar membrane
green
tpFNR:mEosFP x GFP:Vac
yellow,
Mitochondrial
tpFNR:mEosFP x GFP:MAP4
Plastid stroma green to red
convertible, Microtubules green
tpFNR:mEosFP
GFP:mTalin
Plastid
stroma
green
to
photoconvertible,
Actin cytoskeleton green
Tonoplast green, ER lumen red
x
GFP:Vac-T-RFP:ER
photo-
red
Schattat
et
al.2011; Sinclair
et al., 2009
Marques et al.,
2004; Dhanoa
et al., 2010
Schattat et al.,
2011a; Logan &
Leaver, 2000
Schattat et al.,
2012b; Mano et
al., 2002
Schattat et al.,
2012b; Line Q5
from Cutler et
al., 2000
Schattat et al.,
2012b; Mathur
& Chua, 2000
Schattat et al.,
2012b; Kost et
al., 1998
Cutler et al.,
2000; Sinclair et
al., 2009
Immutans (im)-T-tpFNR:GFP
Plastid stroma green
Glabra2 (gl2)-T-tpFNR:GFP
Plastid stroma green
Crooked (crk)
Wurm (wrm)
Wetzel et al,
1994; Marques
et al., 2004
Rerie et al.,
1994; Marques
et al., 2004
Mathur, 2005
Mathur, 2005
Journal of Cell Science • Advance article
Mutant backgrounds
J. Cell Sci. 130: doi:10.1242/jcs.202275: Supplementary information
Supplementary Figures
Supplementary Figure 1. The perimeter of PCC plastid bodies and stromules
that were considered for analysis of mitochondria-PCC juxtaposition. The
perimeters did not differ from one another and on average represented equal
Error bars represent the 95% CI for the estimated mean (N=38).
Journal of Cell Science • Supplementary information
proportions of the exposed plastid surface area in these time-lapsed image sets.
J. Cell Sci. 130: doi:10.1242/jcs.202275: Supplementary information
Supplementary Movies
All time-lapse movies are being played at 5 frames per second.
M1.
Chlorophyll containing plastids (false coloured in magenta) are readily
observed in a pavement cell of the cotyledon petiole that has curled out of the
M2.
A pavement cell in the double transgenic tpFNR:mEosFP-GFP:mTalin line
showed PCC alignment and motility along a F-actin strand.
Journal of Cell Science • Supplementary information
general epidermal plane in the Arabidopsis wurm mutant.
J. Cell Sci. 130: doi:10.1242/jcs.202275: Supplementary information
M3. Time-lapse sequence of a top down view of a pavement cell in a
tpFNR:mEosFP-GFP:Vac line showing a single PCC with an extended stromule
and the vacuolar tubules and vesicles in the neighbourhood. In this view the PCC
M4.
A lateral view showing several PCC in a tpFNR:mEosFP-GFP:Vac line
being engulfed by dynamic vacuolar tubules that appear to flow over them.
Journal of Cell Science • Supplementary information
appears to lie above the vacuoles.
J. Cell Sci. 130: doi:10.1242/jcs.202275: Supplementary information
M5.
A nucleus with several PCC, some with extended stromules around it.
M6.
Time-lapse sequence from a tpFNR:YFP-Mito:GFP line showing the
general movement and occasional stoppings of mitochondria, suggestive of
interactions, around the body and extended stromules of a PCC.
Journal of Cell Science • Supplementary information
Note that the stromules are not always extended towards the nucleus.