Download GFP-labelled Rubisco and aspartate aminotransferase are present

Journal of Experimental Botany, Vol. 55, No. 397, pp. 595±604, March 2004
DOI: 10.1093/jxb/erh062 Advance Access publication 30 January, 2004
RESEARCH PAPER
GFP-labelled Rubisco and aspartate aminotransferase are
present in plastid stromules and traf®c between plastids
Ernest Y. Kwok and Maureen R. Hanson*
Department of Molecular Biology and Genetics, 321 Biotechnology Building, Cornell University, Ithaca, NY 14853,
USA
Received 4 September 2003; Accepted 10 November 2003
Abstract
Plastid stromules are membrane-bound protrusions
of the plastid envelope that contain soluble stroma.
Stromules are often found connecting plastids within
a cell and ¯uorescence recovery after photobleaching
(FRAP) experiments have demonstrated that green
¯uorescent protein (GFP) can move between plastids
via these connections. In this report, the ability of
endogenous plastid proteins to travel through stromules was investigated. The motility of GFP-labelled
plastid aspartate aminotransferase and the Rubisco
small subunit was studied in stromules by FRAP.
Both fusion proteins assemble into protein complexes that appear to behave similarly to their endogenous counterparts. In addition, both enzymes are
capable of traf®cking between plastids via stromules.
Key words: Aspartate aminotransferase, chloroplast, FRAP,
photobleaching, plastid, protein traf®cking, Rubisco,
stromule.
Introduction
Plastid stromules, or stroma-®lled tubules, are tubular
projections of the plastid envelope membrane. Stromulelike structures were recognized as early as the beginning of
the twentieth century, and there have been sporadic
observations since then, but a clear understanding of
their structure or function has never been achieved (KoÈhler
et al., 1997; Gray et al., 2001). Recently, the use of green
¯uorescent protein (GFP) targeted to plastids has facilitated the study of stromules in vivo and in non-green
tissues. Photobleaching of GFP in plastids connected by
stromules has shown that GFP can move between plastids
through stromules (KoÈhler et al., 1997). This discovery of
macromolecular movement between plastids has sparked
new interest in stromules, which have now been observed
in a number of higher plant species by ¯uorescence,
transmitted light, and electron microscopy (Bourett et al.,
1999; Tirlapur et al., 1999; Langeveld et al., 2000; Pyke
and Howells, 2002).
Stromules are highly variable in appearance. Their
length can range from the short projections of 1±2 mm seen
in chloroplasts to extensions up to 50 mm long seen in some
non-green tissues (Bourett et al., 1999; Arimura et al.,
2001). Stromules also show a wide range of diameters,
often in the same cell. Widths have been reported ranging
from less than 100 nm to greater than 1 mm (Langeveld
et al., 2000; Pyke and Howells, 2002). When viewed
in vivo, stromules are highly dynamic structures, extending
and contracting from plastid bodies and moving through
the cytoplasm (Wildman et al., 1962; Kwok and Hanson,
2003). Experiments with inhibitors of the cytoskeleton
have demonstrated that microtubules and actin micro®laments both contribute to the morphology of stromules
(Kwok and Hanson, 2003). Stromule motility, similar to
chloroplast movement, is driven by actin micro®laments,
with an inhibitory contribution from microtubules (Gray
et al., 2001; Kwok and Hanson, 2003).
To date, research has not revealed any function for
stromules in plants. However, ¯uorescence recovery after
photobleaching (FRAP) experiments have shown that GFP
residing in plastids can move through stromules between
plastids (KoÈhler et al., 1997). The motility of a foreign
protein such as GFP (molecular mass=30 kDa) indicates
that other small proteins and metabolites should move
* To whom correspondence should be addressed. Fax: +1 607 255 6249. E-mail: [email protected]
Abbreviations: AAT, aspartate aminotransferase; AAT3, plastid aspartate aminotransferase; CFP, cyan ¯uorescent protein; FRAP, ¯uorescence recovery
after photobleaching; GFP, green ¯uorescent protein; LS, large subunit of Rubisco; L8S8, Rubisco holoenzyme; NPC, nuclear pore complex; Rubisco,
ribulose-1,5-bisphosphate carboxylase/oxygenase; SDS-PAGE, denaturing polyacrylamide gel electrophoresis; SS, small subunit of Rubisco.
Journal of Experimental Botany, Vol. 55, No. 397, ã Society for Experimental Biology 2004; all rights reserved
596 Kwok and Hanson
freely through stromules as well. Furthermore, the movement of GFP suggests that macromolecules like DNA and
RNA might also traf®c. Stromules may, therefore, function
as pathways for movement of metabolites and macromolecules between plastids in different parts of the cell.
Hence, stromules may possibly be considered as organellar
analogues of plasmodesmata, the cytoplasmic connections
that join plant cells in many tissues.
Macromolecular movement through plasmodesmata is
believed to occur via 2.5 nm channels in the cytoplasmic
sleeve (Ding et al., 1992). Plasmodesmata are characterized by two forms of traf®cking. A non-regulated
traf®cking, de®ned by the basal size exclusion limit,
allows the diffusion of molecules ranging in size from less
than one kDa up to 60 kDa, depending on tissue and
developmental state (reviewed in Zambryski and
Crawford, 2000). Regulated movement is observed for
larger proteins and nucleic acids (Zambryski and
Crawford, 2000). Parallels can also be drawn between
stromules and the nuclear pore complex (NPC). The NPC
is a collection of more than 100 proteins residing in the
nuclear envelope that regulates traf®c between the nucleus
and cytoplasm. The NPC is a cylindrical pore with a
diameter of 120 nm and a height of 70 nm (reviewed in
Heese-Peck and Raikhel, 1998). NPCs also participate in
two forms of traf®cking. One system allows the free
diffusion of metabolites and small macromolecules of up
to 30 kDa, probably by a 9 nm channel (Paine et al., 1975).
The second system utilizes a 26 nm regulated channel for
energy-dependent import and export of proteins, RNA, and
very large ribonucleoproteins of molecular mass up to 10
MDa (GoÈrlich and Kutay, 1999).
The traf®cking capacities of plasmodesmata and the
NPC have been determined with some precision. By
contrast, the nature of molecular movement in stromules is
still unknown. As a ®rst step in dissecting protein
traf®cking in stromules, experiments were conducted to
determine if, in addition to GFP, endogenous plastid
proteins can traf®c via stromules, and if protein traf®cking
is limited by protein size. Two previous reports describe
the presence of endogenous proteins in stromules. Tirlapur
et al. (1999) fused two-thirds of the PAC protein to GFP
and expressed the fusion in Arabidopsis thaliana. They
found that GFP ¯uorescence was present in stromules and
that the GFP could traf®c between plastids. Bourett et al.
(1999) localized the Rubisco large subunit (LS) to short
projections of chloroplasts in Oryza sativa by immunocytochemistry. However, to the authors' knowledge, no
group has reported traf®cking of full length endogenous
plastid proteins or protein complexes.
In the work described below, GFP was fused to
endogenous plastid proteins and their motility in stromules
was observed by a photobleaching method. Two proteins
were selected for study: the plastid aspartate aminotransferase (AAT3) and the Rubisco small subunit (SS). Both
are nuclear-encoded proteins that are translated on
cytosolic ribosomes and subsequently imported into
plastids. Aspartate aminotransferases (AAT) are highly
conserved enzymes found across kingdoms. These 45 kDa
proteins are active as 90 kDa homodimers. AAT enzymes
catalyse the reversible formation of oxaloacetate and
glutamate from aspartate and a-ketoglutarate using
pyridoxal-5¢-phosphate as a cofactor (reviewed in
Wadsworth, 1997). In plants, AAT is believed to have
roles in amino acid synthesis, ammonia assimilation,
intercellular carbon shuttling in C4 plants, and interorganellar hydrogen shuttling. Higher plants contain multiple
forms of AAT, specialized for function in various
compartments in the cell. The plastid-localized enzyme,
AAT3, was the focus of attention in this work.
Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) is the major protein constituent of chloroplasts
(reviewed in Gutteridge and Gatenby, 1995). This enzyme
functions in photosynthetic carbon ®xation and photorespiratory oxygen ®xation. Rubisco is a 550 kDa enzyme
composed of 16 subunits. Eight 14.5 kDa small subunits
(SS) combine with eight 55 kDa large subunits (LS) to
form the holoenzyme (L8S8).
90 kDa AAT3 and 550 kDa Rubisco therefore provided
a convenient size range to assay for the presence in
stromules and traf®cking between plastids. Protein assembly experiments con®rm that the fusion proteins that were
generated behave similarly to their endogenous counterparts in transgenic plants. Furthermore, FRAP assays
demonstrate that both fusion proteins are capable of
moving through stromules.
Materials and methods
Fusion protein constructs
Standard molecular biology techniques were used for all experiments. Unless otherwise noted, all chemicals were obtained from
Sigma (St Louis, MO, USA).
Constructs for plant transformation were generated in a pGPTV
binary vector (Becker et al., 1992). For the ASP5:GFP construct, a
cDNA of the A. thaliana ASP5 gene was used as a template for PCR
ampli®cation. The forward primer contained an NcoI restriction site
at its 5¢ end. The reverse primer included sequences encoding a ®ve
amino acid linker (GSGGG) at its 3¢ end to promote proper folding
and activity of ASP5 and GFP (Chandler et al., 1998).
mGFP5(S65T) was ampli®ed by PCR using a forward primer that
overlapped the ASP5 fragment and a reverse primer with an SstI site
at the 5¢ end. These two fragments were joined via recombinant
PCR, sequenced, and inserted into the binary vector. For the SS:CFP
construct, a cDNA of the Pisum sativum RbcS-3A gene, including the
native plastid transit peptide, was used for PCR ampli®cation. The
forward primer contained an XbaI restriction site at the 5¢ end while
the reverse primer contained a BamHI site at its 5¢ end. mCFP was
ampli®ed using a forward primer with a BamHI site and sequences
encoding the same ®ve amino acid linker used for ASP5:GFP at its 5¢
end. The reverse primer contained an SstI site at its 5¢ end. These two
fragments were joined by restriction digestion and ligation,
sequenced, and inserted into the binary vector. For the TP:CFP
construct, the RbcS-3A transit peptide sequence was ampli®ed using
Protein traf®cking in stromules 597
a forward primer with an XbaI site at its 5¢ end and a reverse primer
that overlapped with mCFP. mCFP was ampli®ed with a forward
primer and a reverse primer identical to the reverse primer used for
CFP in the SS:CFP construct. The two fragments were joined by
recombinant PCR, sequenced, and inserted into the binary vector.
Agrobacterium-mediated transformation
Leaf strips of Nicotiana tabacum cv. Samsun NN were transformed
with Agrobacterium tumefaciens strain LBA4404 as described by
Horsch et al. (1985). Transgenic cells were selected on MS104
medium (2.2 g l±1 MS basal medium with Gamborg's vitamins, 1 mg
l±1 6-benzylaminopurine (BA), 0.1 mg l±1 a-naphthaleneacetic acid
(NAA), 30 g l±1 sucrose, and 8 g l±1 phytagar) supplemented with 300
mg ml±1 kanamycin and 100 mg ml±1 ticarcillin/clavulanic acid (15:1;
Duchefa, Haarlem, The Netherlands). Regenerated plants were
transferred to soil and screened for ¯uorescence and GFP protein by
western blot (data not shown). Callus cultures were generated from
leaf strips on solid NT1 medium (4.33 g l±1 MS basal salt mixture,
100 mg l±1 myo-inositol, 1 mg l±1 thiamine, 0.2 mg l±1 2,4dichlorophenoxyacetic acid, 180 mg l±1 KH2PO4, and 9 g l±1
phytagar). Callus cells were placed in ¯asks of liquid NT1 and
agitated at 110 rpm to induce suspension cell growth. Suspension
cultures were agitated in liquid NT1 at room temperature in the dark
and transferred weekly.
Microscopy
Laser scanning confocal microscopy was performed with a Leica
TCS-SP2 confocal scanning head mounted on a Leica DMRE-7
(SDK) upright microscope equipped with a 1003 HCX PlAPO oil
immersion objective (NA=1.40; Leica Microsystems Inc.,
Bannockburn, IL, USA). GFP was excited with the 488 nm line of
a 4-line Argon ion laser and emission was detected by collecting
light between 500 and 600 nm. CFP was excited at 458 nm and
emission was collected between 500 and 600 nm. Chlorophyll was
excited at 633 nm and emission was detected between 660 and 700
nm. DIC images were generated by collecting transmitted light. For
each picture, several images were collected at successive focal
planes along the z-axis and then reassembled as a maximum
projection.
Protein analysis
Proteins for AAT activity assay were isolated by grinding plant
tissues in an extraction buffer described by Sentoku et al. (2000).
Soluble chloroplast proteins were isolated as described by Gruissem
et al. (1986). Root tissue was obtained from plants grown on liquid
MS medium (2.2 g l±1 MS basal medium with Gamborg's vitamins,
30 g l±1 sucrose) for 2±3 weeks. Proteins were separated on nondenaturing polyacrylamide gels (PAGE) as described by Schultz and
Coruzzi (1995). AAT activity was detected by in-gel assay as
described by Wendel and Weeden (1989). Brie¯y, gels were soaked
for 30 min at room temperature with gentle agitation in a solution
containing 2.5 mM a-ketoglutaric acid, 10 mM L-aspartic acid, 125
mM PVP-40, 1.34 mM Na2EDTA (Fisher Scienti®c, Fair Lawn, NJ,
USA), 100 mM dibasic sodium phosphate (Fisher Scienti®c), and 1
mg ml±1 Fast Blue BB salt. GFP ¯uorescence in AAT activity bands
was analysed on a Storm 840 phosphorimager using 450 nm
excitation (Amersham Biosciences, Piscataway, NJ, USA).
Tissues for immunoblot analysis were frozen in liquid nitrogen
and ground in buffer containing 0.1 M TRIS-HCl pH 7.5, 0.1 M
NaCl, 5 mM EDTA, 10 mM 2-mercaptoethanol, and 2.5 mM
phenylmethylsulphonyl¯uoride. Proteins in extracts were quanti®ed
by Bradford assay and boiled for 5 min in loading buffer (250 mM
TRIS-HCl pH 6.8, 20% glycerol, 4% sodium dodecyl sulphate, 2%
2-mercaptoethanol, and a trace of bromophenol blue). Proteins were
separated on 12% SDS-PAGE and transferred to nitrocellulose
membranes. For the detection of Rubisco, membranes were blocked
in TBS-T (8 g l±1 NaCl, 0.2 g l±1 KCl, 3 g l±1 TRIS, and 0.05%
Tween-20) containing 5% (w/v) powdered milk. A polyclonal
antibody was used to detect Rubisco LS and SS. For detection of
GFP and CFP, membranes were blocked in TSW (10 mM TRIS, 9 g
l±1 NaCl, 2.5 g l±1 gelatin, 0.1% Triton X-100, 0.2 g l±1 SDS).
Fluorescent proteins were detected by a monoclonal antibody that
detects both GFP and CFP. Antibody binding was detected by a
secondary antibody conjugated to horseradish peroxidase and
enzyme activity detected by chemiluminescence.
Proteins for Rubisco assembly analysis were extracted by grinding
in non-denaturing buffer (62 mM TRIS-HCl pH 7.0, 0.5% 2mercaptoethanol). Soluble proteins were separated by PAGE on 4%
gels. GFP ¯uorescence was detected using a phosphorimager as
described above.
Fluorescence recovery after photobleaching
FRAP was performed using the Leica confocal microscope
described above. For each experiment, a suitable plastid pair was
selected and a pre-bleach image acquired at 10% laser power and 43
zoom. Bleaching was accomplished with a single scan by using
100% laser power and zooming into the bleach area at 323 zoom.
Recovery images were collected at the same resolution and laser
power as the pre-bleach image at 1.6 s intervals for 24 s and then at
10 s intervals for 60 s. 12-bit images were analysed using Leica TCS
software. Total ¯uorescence was calculated in the regions of interest
around the plastids in question and expressed as relative ¯uorescence
by dividing by each plastid's ¯uorescence in the pre-bleach image.
Results
GFP fusion proteins for monitoring protein traf®cking
To study the movement of plastid proteins in stromules,
two nuclear-encoded plastid proteins of different sizes
were selected for fusion to ¯uorescent proteins: the major
plastid-localized aspartate aminotransferase and the
Rubisco small subunit. In A. thaliana, the ASP5 gene
encodes AAT3 activity (Wilkie et al., 1996; Schultz et al.,
1998). Rubisco SS is coded in the nucleus by a small gene
family in most plants, including N. tabacum (Jamet et al.,
1991).
Transgenic N. tabacum plants were generated using a
pGPTV binary vector that carries a kanamycin resistance
marker for selection in plants (Becker et al., 1992). Coding
regions for fusion proteins were inserted downstream of a
double cauli¯ower mosaic virus 35S promoter and alfalfa
mosaic virus translation enhancer (Datla et al., 1993). The
coding regions were followed by a nopaline synthase 3¢
untranslated region (Becker et al., 1992). Constructs were
introduced into N. tabacum cv. Samsun NN via
Agrobacterium-mediated transformation of leaf strips.
Constructs for plant transformation are shown in Fig. 1.
The aspartate aminotransferase construct consisted of the
A. thaliana ASP5 coding region (Wilkie et al., 1995)
followed by mGFP5(S65T) (Siemering et al., 1996). This
construct was designated ASP5:GFP. GFP was fused to the
carboxy-terminus of ASP5 to preserve the cleavage site
between the ASP5 transit peptide and mature protein, thus
ensuring proper plastid import and processing. A ®ve
598 Kwok and Hanson
stromules was examined. Fusion proteins were also tested
for their ability to assemble into the expected higher order
molecular complexes.
Endogenous AAT3 expression and assembly of
ASP5:GFP
Fig. 1. Constructs for stable nuclear transformation of N. tabacum.
Constructs were inserted into binary vectors between a double-CaMV
35S promoter and nopaline synthase 3¢ UTR. ASP5: A. thaliana plastid
aspartate aminotransferase. GFP: green ¯uorescent protein. SS: P.
sativum small subunit of Rubisco. CFP: cyan ¯uorescent protein. TP:
plastid transit peptide. Asp5:GFP contains the A. thaliana ASP5 transit
peptide. SS:CFP and TP:CFP contain the P. sativum SS transit peptide.
Sizes of mature proteins are given in amino acids under each label.
amino acid linker peptide (GSGGG) was inserted between
ASP5 and GFP to promote proper folding and activity of
both proteins. The Rubisco small subunit construct consisted of the rbcS-3A coding region of P. sativum (Fluhr
et al., 1986) followed by cyan ¯uorescent protein (mCFP)
(Haseloff, 1999). This construct was designated SS:CFP.
The rbcS-3A coding region includes a plastid transit
peptide sequence. As with ASP5:GFP, a ®ve amino acid
linker peptide was used to join SS and CFP. A control
construct in which CFP alone was targeted to plastids via
the rbcS-3A transit peptide was also made. This construct
was designated TP:CFP.
Expression of fusion proteins in transgenic plants
Stable transgenic lines that showed both high and low
levels of ¯uorescence were regenerated for each construct.
None of the plants showed any obvious changes in growth
or morphology relative to the wild type when grown on soil
or in culture. In all transgenic lines, ¯uorescence was
detected speci®cally in chloroplasts and non-green plastids
(Fig. 2). Epi¯uorescence and confocal ¯uorescence
microscopy also revealed ¯uorescence in plastid stromules
in all tissues and organs known to have high frequencies of
stromules, namely: hypocotyl epidermis, cultured suspension cells, ¯ower petals, and root epidermis and hair cells
(Fig. 2). Thus ASP5:GFP and SS:CFP are present in
stromules of N. tabacum. In most transgenic lines, an even
¯uorescence signal was observed in all tissues and organs.
The single exception was found in roots of SS:CFP plants.
SS:CFP ¯uorescence in roots was extremely low relative to
the ¯uorescence signal seen in other parts of the same
transgenic plant (Fig. 2D). This difference in ¯uorescence
may be due to post-translational regulation of Rubisco
subunits (see Discussion).
Before testing the fusion proteins for stromule traf®cking, it was necessary to assess whether the fusion proteins
behaved as their endogenous counterparts and could
therefore serve as indicators of native protein activity in
stromules. To this end, wild-type expression of the two
proteins in N. tabacum tissues and organs that are rich in
Aspartate aminotransferase activity assays were performed
on untransformed N. tabacum to determine the distribution
of AAT3 expression (Fig. 3). In A. thaliana, AAT activity
resolves into three distinct bands on native PAGE (Schultz
and Coruzzi, 1995). The plastid isozyme, AAT3, migrates
fastest on these gels, with mitochondrial and cytosolic
AAT running signi®cantly slower (Schultz and Coruzzi,
1995). A similar pattern was observed in N. tabacum
(Fig. 3). Although it was not always possible to resolve the
mitochondrial from the cytosolic bands, a fast-migrating
band speci®c to chloroplasts was visible. Analysis of
extracts from various organs and tissues con®rmed that N.
tabacum plastid AAT is present in all tissues that carry
high numbers of stromules (Fig. 3).
AAT3, like all other plant aspartate aminotransferases,
assembles into homodimers in vivo (Wilkie et al., 1996).
Monomeric AAT has been demonstrated to be nonfunctional in vitro (Arrio-Dupont and Coulet, 1979). To
investigate the assembly of ASP5:GFP fusion proteins,
protein extracts of transgenic plants were assayed for AAT
activity and GFP ¯uorescence (Fig. 4). Non-denaturing
PAGE gels stained for AAT activity indicate that
ASP5:GFP is able to dimerize with itself and also forms
heterodimers with the endogenous N. tabacum AAT3
(Fig. 4A). Extracts from transgenic plants contain two
additional AAT activity bands that migrate between the
endogenous plastid and cytosolic/mitochondrial bands.
The presence of two additional activity bands indicates the
fusion protein is able to form dimers with itself (giving rise
to the slower moving band) and also with the endogenous
AAT3 protein (giving rise to the faster moving band) to
give the functional enzyme. Comparison of activity
staining in the wild type (Fig. 3) and ASP5:GFP (Fig. 4)
shows a relative loss of endogenous plastid AAT activity
with respect to cytosolic and mitochondrial AAT activity
in the ASP5:GFP plants. This is due to the diversion of
some of the endogenous plastid AAT into the formation of
heterodimers with ASP5:GFP fusions. Fluorescence analysis of the activity gels con®rmed the presence of GFP in the
bands containing fusion proteins (Fig. 4B). ASP5:GFP
homodimers and heterodimers were found in all stromulebearing organs and tissues. Thus A. thaliana ASP5, with
GFP fused to its carboxy-terminus, can interact with itself
and with the N. tabacum AAT3 to form enzymes that are
functional in vitro.
Endogenous SS expression and assembly of SS:CFP
High level expression of Rubisco SS is normally limited to
chloroplasts in green tissue, as expected for a protein
Protein traf®cking in stromules 599
Fig. 2. ASP5:GFP and SS:CFP accumulate in stromules of transgenic N. tabacum. Confocal ¯uorescence micrographs of GFP and CFP
¯uorescence in N. tabacum expressing SS:CFP (A±F) and ASP5:GFP (G±L). In both lines, ¯uorescence from fusion proteins overlapped with
chlorophyll auto¯uorescence in leaf chloroplasts, con®rming correct plastid localization (A, G). Fluorescence was also detected in plastid
stromules in light-grown hypocotyl epidermis (B, H), petal epidermis from pink region of corolla (C, I), root hairs (D, J), dark-grown hypocotyl
epidermis (E, K), and liquid-cultured suspension cells (F, L). In each image, GFP/CFP ¯uorescence is pseudocoloured green, chlorophyll
¯uorescence is pseudocoloured red, and differential interference contrast (Nomarski) images are pseudocoloured blue. Images are maximum
projections of several confocal images taken along the z-axis. Bars: 5 mm.
involved in photosynthesis (Tobin and Silverthorne, 1985).
Experiments on N. tabacum indicate that both LS and SS
accumulate to low levels in ¯ower petals and dark-grown
seedlings (Fig. 5). Protein extracts of wild-type N. tabacum
were separated by SDS-PAGE and blotted to nitrocellulose. Immunodetection with a polyclonal antibody directed
against Rubisco detected both LS and SS in leaf, petal, and
dark-grown hypocotyls. Rubisco was undetectable in lightgrown roots and suspension cells (Fig. 5).
SS normally forms a 550 kDa holoenzyme with plastidencoded LS. In SS:CFP transgenic plants, the SS:CFP
fusion assembles into the 16-subunit holoenzyme in leaves
and dark-grown seedlings of transgenic plants (Fig. 6).
Extracts of leaf proteins were separated by non-denaturing
PAGE, preserving the holoenzyme assembly of Rubisco
(Fig. 6A). Transgenic protein extracts from leaves and
dark-grown seedlings contain a single slow-migrating
protein band. SS:CFP transgenic petals contain a similarly
600 Kwok and Hanson
slow-migrating band as well as a signi®cantly faster
migrating ¯uorescent band. Leaf extracts of TP:CFP plants
contain a single fast-migrating protein band. The similar
mobility of the TP:CFP band and the fast-migrating band
of SS:CFP petals suggests this SS:CFP petal protein is
unassembled SS:CFP fusion protein. The fact that SS:CFP
petal extracts contained both assembled SS:CFP and an
unassembled SS:CFP protein made it unsuitable for further
analysis, since the unassembled protein would not accurately represent endogenous Rubisco. Wild-type leaf
extracts showed no ¯uorescent protein bands, as expected.
To identify the proteins contained in the slow-migrating
¯uorescent bands, the ¯uorescent bands from leaf extracts
of SS:CFP, and TP:CFP, as well as the gel region in the
wild-type sample at the same position as the SS:CFP band
were cut from the non-denaturing gels (white boxes in
Fig. 6A) and introduced into the lanes of SDS-PAGE gels
(Fig. 6B). Denatured and separated proteins from the gel
slices were probed with antibodies for Rubisco (Fig. 6B).
Wild-type samples contained both LS and SS, con®rming
Fig. 3. Endogenous plastid aspartate aminotransferase is expressed in
all stromule-rich tissues. Protein extracts from isolated chloroplasts
and tissues of untransformed N. tabacum were separated by nondenaturing PAGE and stained for AAT activity. Protein extract from
puri®ed chloroplasts (Cp) contains a single fast-migrating AAT
activity band (AAT3). Protein extract from mature leaves (Lf), lightgrown roots (Rt), pink regions of ¯ower petals (Pt), whole dark-grown
seedlings (Dk), and cultured suspension cells (SC) contain both the
plastid AAT3 as well as a slower migrating cytosolic and
mitochondrial AAT activity band (Cyt/Mt). 15 mg total protein was
loaded for each sample.
that the gel slices excised from native gels in Fig. 6A
contained the Rubisco holoenzyme and indicating that
SS:CFP fusion protein forms a complex that migrates
identically to the endogenous Rubisco holoenzyme. This
was con®rmed by the presence of LS and SS in the SS:CFP
sample. The SS:CFP sample also contained an additional
band migrating slightly faster than LS that corresponds to
the predicted size of the SS:CFP fusion protein. Thus the
¯uorescence in SS:CFP plants is due to SS:CFP fusion
protein that is assembled with endogenous SS and LS to
form the Rubisco holoenzyme. The TP:CFP sample
contained no Rubisco protein, con®rming that CFP by
itself does not interact with Rubisco SS or LS.
Traf®cking of fusion proteins
The above experiments con®rmed that ASP5:GFP and
SS:CFP assembled into their respective complexes and
could accumulate in stromules. It was next determined
Fig. 5. Rubisco is expressed in leaves, petals, and dark-grown
seedlings of wild-type N. tabacum. Total soluble protein from wildtype plants was separated by SDS-PAGE. Gels were blotted to
membranes and probed with antibodies recognizing both subunits of
Rubisco. Rubisco large subunit (LS) and small subunit (SS) were
found in leaves (Lf), petals (Pt), and dark-grown seedlings (Dk).
Neither of the Rubisco subunits was found in roots (Rt) or suspension
cells (SC). Protein size markers indicate migration positions of
proteins in kDa. Extracts were loaded in the following amounts: leaf,
2 mg; root, 15 mg; petal, 15 mg; cell, 30 mg; dark, 7.5 mg.
Fig. 4. ASP5:GFP assembles into homodimers and heterodimers in transgenic N. tabacum. Protein extracts from wild-type and ASP5:GFP
transgenic plants were separated by non-denaturing PAGE to preserve protein complex structure. (A) AAT activity staining of ASP5:GFP extracts
showed endogenous plastid and cytosolic/mitochondrial AAT activity bands (AAT3/AAT3 and Cyt/Mt, respectively) as well as two activity bands
that are not normally found in wild-type N. tabacum: homodimers of ASP5:GFP (ASP5:GFP/ASP5:GFP), and heterodimers between ASP5:GFP
and the endogenous plastid-localized AAT (ASP5:GFP/AAT3). (B) Fluorescence analysis of the gel in (A) con®rms that GFP is present in both
the homodimer and heterodimer bands. WT: wild-type N. tabacum. ASP5:GFP: transgenic N. tabacum expressing ASP5:GFP fusion. Cp: soluble
fraction of puri®ed chloroplasts. Lf: mature leaves. Rt: light-grown roots. Pt: pink region of ¯ower petals. Dk: dark-grown seedlings. SC: liquidcultured suspension cells. 20 mg total protein was loaded for each sample except for WT Cp, and ASP5:GFP Lf and Pt, which contained
10 mg protein each.
Protein traf®cking in stromules 601
whether these fusion proteins could also traf®c between
plastids via stromules, as had been demonstrated for
unfused GFP. Fluorescence recovery after photobleaching
was used to monitor the movement of the fusion proteins
between plastids in dark-grown hypocotyls. FRAP was
conducted on dark-grown hypocotyls because stromules
are numerous in these organs and often connect pairs of
plastids. In addition, both AAT3 and Rubisco are normally
expressed in these cells and the fusion proteins had been
shown to assemble correctly here. When plastids containing SS:CFP or ASP5:GFP were bleached of ¯uorescence,
¯uorescence gradually recovered, at the expense of
¯uorescence in a connected plastid (Fig. 7). Recovery
was generally complete within the 2 min observation
period. Control experiments in which a single plastid
without stromules was bleached and then monitored over
Fig. 6. SS:CFP assembles into Rubisco holoenzyme in leaves and
dark-grown hypocotyls of transgenic N. tabacum. Fluorescent protein
fractions of SS:CFP plants were analysed for presence of Rubisco
subunits. (A) Non-denaturing PAGE scanned for CFP ¯uorescence.
SS:CFP protein extracts from leaves (Lf), pink ¯ower petals (Pt), and
dark-grown seedlings (Dk) contain slow-migrating ¯uorescent protein
bands (L8S8). The petal sample also contains a fast-migrating band
(SS:CFP). TP:CFP leaf extract contains only a fast-migrating band
(TP:CFP). White squares indicate regions of the gel that were excised
and loaded onto SDS-PAGE gels shown in (B). (B) Proteins from gel
slices in (A) were separated by SDS-PAGE and Rubisco protein was
detected by antibodies. Wild-type gel slice contains both Rubisco LS
and SS. SS:CFP gel slice contains Rubisco LS and SS as well as
SS:CFP fusion protein (arrow). TP:CFP gel slice contains no Rubisco
proteins. Size markers indicate relative mobility of proteins in kDa.
time showed no signi®cant recovery of ¯uorescence (data
not shown), con®rming that bleaching was due to the
irreversible destruction of the GFP or CFP ¯uorophore
(Kwok and Hanson, 2003). Likewise, unbleached plastids
that were monitored over time showed no signi®cant loss
of ¯uorescence, con®rming that ¯uorescence is not lost by
photobleaching under these experimental conditions. The
observation of ¯uorescence recovery for the fusions in
hypocotyl epidermal cells demonstrates that SS:CFP and
ASP5:GFP can traf®c between plastids via stromules.
Discussion
FRAP experiments (Fig. 7) showed that fusion proteins of
¯uorescent proteins and the endogenous plastid proteins
aspartate aminotransferase and Rubisco small subunit are
able to traf®c through stromules. The fusion proteins
introduced into N. tabacum plastids behaved identically to
their endogenous counterparts in terms of assembly into
multimeric complexes. The traf®cking data from fusion
proteins can therefore be extended to the endogenous N.
tabacum proteins. Thus, 90 kDa plastid AAT dimers and
550 kDa Rubisco holoenzymes are motile in plastid
stromules.
Plastid aspartate aminotransferase is believed to have a
number of metabolic activities in the cell and is therefore
expressed throughout the plant body (Fig. 3). The
ASP5:GFP fusion protein was able to assemble with itself
and with N. tabacum AAT3 in vivo and both of these
dimers were functional in vitro (Fig. 4). Therefore, the
fusion protein provides an accurate tool for studying
endogenous AAT3 motility. In fact, because the fusions
are larger than endogenous AAT3 because of the extra 30
kDa associated with the fused GFP, the FRAP experiments
underestimate the motility of endogenous AAT3.
Rubisco, with its 550 kDa size and its critical role in
photosynthesis is a compelling target for study. However,
the fact that it is not expressed to a high degree in nongreen tissues, where stromules are most frequently found,
limited its analysis. Expression analysis showed that
Rubisco accumulated to signi®cant levels only in leaves,
dark-grown seedlings, and ¯ower petals, with no protein
accumulating in roots or suspension cells (Fig. 5). Coruzzi
et al. (1984) reported similar ®ndings in P. sativum: the
Rubisco small subunit protein accumulated to high levels
in leaves and petals. However, these authors found that SS
protein accumulation in dark-grown P. sativum seedlings
was less than 1% of leaf levels, though protein was
detected at a high level in seeds, and rbcS mRNA was
detected in dark-grown seedlings (Coruzzi et al., 1984).
Expression studies of Rubisco in wild-type N. tabacum
showed no protein accumulation in suspension cells or
roots (Fig. 5). However, ¯uorescence was detected in
suspension cells and roots of transgenic plants (Fig. 2),
suggesting the SS:CFP fusion was stable in the absence of
602 Kwok and Hanson
Fig. 7. ASP5:GFP and SS:CFP fusion proteins move between plastids via stromules. FRAP experiments were conducted in dark-grown hypocotyl
epidermal cells of transgenic N. tabacum. Images and quantitative data are provided for FRAP experiments on TP:CFP (A±D), ASP5:GFP (E±H),
and SS:CFP (I±L). (B, F, J) Prebleach images. Bl: bleached plastid. Un: unbleached plastid. (C, G, K) Postbleach images. (D, H, L) Recovered
images. Fluorescence intensity of bleached plastids, unbleached plastids, and unconnected control plastids are shown in (A, E, I), represented by
open circles, closed triangles and open squares, respectively. The ¯uorescence at each time point is normalized to the ¯uorescence at the
beginning of the experiment.
other Rubisco subunits. This is unexpected, as the
accumulation of SS and LS is tightly regulated to maintain
stoichiometric amounts of both proteins in the plastid
stroma (Rodermel, 1999). In particular, Schmidt and
Mishkind (1983) studied SS accumulation in
Chlamydomonas reinhardtii in which LS was not expressed because of inhibition of plastid translation. They
found that SS protein was translated and imported into
chloroplasts, but was then rapidly degraded. These data
predict that SS:CFP should be rapidly degraded in root
cells, where no LS is expressed. However, it should be
noted that ¯uorescence in roots of SS:CFP plants was
extremely low relative to all other tissues of the plant,
suggesting post-import degradation did occur to some
extent. Suspension cells also showed high levels of
¯uorescence, suggesting a lack of regulation in these
cells. Similarly, in ¯ower petals, unassembled SS:CFP was
detected in addition to Rubisco holoenzyme (Fig. 6). This
is another instance where the regular subunit control is
violated in SS:CFP transgenic plants. Dark-grown hypocotyls were the only organ outside of leaves where
endogenous Rubisco was expressed and where SS:CFP
fusions assembled correctly. FRAP experiments on these
cells showed traf®cking of the fusion proteins.
Enzymatic activity of hybrid Rubisco enzymes formed
by the combination of endogenous LS and a mixture of
endogenous SS and SS:CFP was not determined in the
transgenic plants. However, recent studies involving the
creation of hybrid forms of Rubisco suggest that Rubisco
subunits can be exchanged and retain function. Kanevski
et al. (1999) replaced N. tabacum LS with LS from
Helianthus annuus (sun¯ower). They found the hybrid
protein to have reduced, but detectable activity. Getzoff
et al. (1998) overexpressed P. sativum rbcS-3a in A.
Protein traf®cking in stromules 603
thaliana and found only a slight reduction in Rubisco
activity. It is therefore possible that the P. sativum RbcS3a, N. tabacum LS hybrids in this study also retained some
level of activity.
In conclusion, stromules represent a signi®cant avenue
of protein movement within the plant cell. The above
experiments indicate that protein complexes as large as
550 kDa can move via stromules. Stromules are thus
another component in the protein traf®cking machinery of
plant cells, with the ability to move macromolecules much
like plasmodesmata and nuclear pore complexes.
Currently, the function of stromules in plants is unknown.
The capacity of stromules for macromolecular traf®cking
suggests stromules may serve as conduits for mixing the
contents of plastids in different parts of the cell. The
presence of enzymes in stromules indicates that stromules
participate in plastid-biochemical activity. The high surface area:volume ratio of stromules may enhance the
exchange of reactants and products between plastids and
the cytoplasm. Future characterization of the molecular
contents of stromules and the mechanism of stromule
transport will shed more light on stromule function.
Acknowledgements
The authors would like to express their gratitude for the material
contributions of several colleagues. The ASP5 coding region of A.
thaliana was provided by Sue Wilkie and Martin J Warren
(University College, London). The rbcS-3a coding region from P.
sativum was contributed by Danny Schnell (University of
Massachusetts, Amherst). Coding regions for mCFP and
mGFP5(S65T) were provided by Jim Haseloff (University of
Cambridge). Hans Bohnert (University of Illinois, UrbanaChampaign) contributed the antibody directed against Rubisco.
This work was supported by Department of Energy grant DE FG0289 ER14030.
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