1 Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla,
CA 92037, USA
2 Department of Biology, University of California San Diego, La Jolla, CA 92093,
USA
3 Department of Plant and Microbial Biology, University of California, Berkeley,
CA 94720, USA
4 Department of Biochemistry and Cell Biology, State University of New York,
Stony Brook, NY 11794-5215, USA
5 Department of Molecular Biology, Max Planck Institute for Developmental
Biology, D-72076 Tübingen, Germany
* Author for correspondence (e-mail: weigel{at}weigelworld.org)
Accepted 2 May 2003
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SUMMARY |
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Key words: Arabidopsis, Protein trafficking, Movement protein, LEAFY, APETALA1
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INTRODUCTION |
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Although plants lack homologs of the well-known metazoan peptide ligands,
such as EGF, TGFß or Hedgehog, plant cells can also communicate via
secreted molecules (Fletcher et al.,
1999; Matsubayashi et al.,
2001
; McCarty and Chory,
2000
). However, in contrast to animals, cytoplasmic continuity
between plant cells is the rule, not the exception. Most plant cells are
connected by plasmodesmata, plasma membrane-lined channels that provide
cytoplasmic continuity between adjacent cells. Plasmodesmata, which are used
in the transport of nutrients and signaling molecules including RNAs and
proteins, can be divided into two major groups
(Crawford and Zambryski, 1999
;
Haywood et al., 2002
;
Lucas, 1995
). The primary
plasmodesmata form during cytokinesis, whereas the secondary plasmodesmata
develop between cells that are not necessarily clonally related. The size
exclusion limit (SEL) of the different types of plasmodesmata can be measured
using fluorescent tracer molecules. In most cases, plasmodesmata in younger
tissues have larger SEL and are morphologically simpler than those in older
tissue (Crawford and Zambryski,
2001
).
Two modes of movement through plasmodesmata have been proposed. Targeted
movement involves specific interactions between the transported macromolecules
and plasmodesmata components. This leads to an increase in the SEL and is
therefore not limited by the endogenous SEL of a given cell. By contrast,
non-targeted movement resembles passive diffusion and is governed by the
endogenous SEL of the plasmodesmata involved
(Crawford and Zambryski, 2000;
Imlau et al., 1999
;
Oparka et al., 1999
). The best
understood case of targeted movement is probably the trafficking of plant
viral movement proteins (MPs), which can move over long distances in plants
and are key to the spreading of plant viral infections. A good example of
non-targeted movement is provided by green fluorescent protein (GFP). Using
transient transfection by bombardment, it has been shown that native GFP can
move several cells away from its source. In addition to the SEL,
multimerization and the addition of nuclear or ER localization signals can
hinder, or even prevent, GFP from leaving the source cell
(Crawford and Zambryski, 2000
;
Crawford and Zambryski,
2001
).
Apart from viral proteins, studies of macromolecule movement in plants have
focused traditionally on long-distance transport of photosynthates and larger
molecules or complexes through the phloem
(Lucas, 1995;
Zambryski and Crawford, 2000
).
A good example is the sucrose transporter SUT1, whose mRNA is transported into
the phloem before it is translated
(Kühn et al., 1997
).
Moreover, grafting experiments have demonstrated the existence of
long-distance mRNA movement in plants (Kim
et al., 2001
; Ruiz-Medrano et
al., 1999
). More recently, studies conducted in several plant
species have demonstrated that non-cell-autonomous effects of transcription
factors involved in plant development can be mediated by protein movement
(reviewed by Haywood et al.,
2002
; Wu et al.,
2002
).
The first example of transcription factor movement was discovered through
studies of the homeodomain protein KNOTTED1 (KN1) in maize. Most plant organs
originate post-embryonically from meristems, which include stem cells set
aside during embryogenesis. In the aerial part of the plant, new organs emerge
from the shoot apical meristem (SAM), which consists of three tissue layers,
L1-L3. KN1 protein is found throughout the maize SAM but kn1 mRNA is
absent from the L1 layer (Jackson et al.,
1994; Smith et al.,
1992
). In leaf injection experiments, not only was KN1 transported
to the surrounding tissue through plasmodesmata, but KN1 also increased the
SEL of plasmodesmata, enabling the transport of kn1 sense RNA and
protein complexes (Kragler et al.,
2000
; Lucas et al.,
1995
). KN1 can also move away from its source of expression when
expressed from heterologous promoters in Arabidopsis
(Kim et al., 2002
). There is
similar evidence that the Antirrhinum MADS-box transcription factor
DEFICIENS (DEF) moves from inner to outer tissue layers in developing flowers,
although the extent of movement is stage- and organ-dependent
(Perbal et al., 1996
).
In Arabidopsis, two endogenous transcription factors move into
neighboring cells: SHORTROOT (SHR)
(Nakajima et al., 2001) and
LEAFY (LFY) (Sessions et al.,
2000
). RNA of the GRAS-family transcription factor SHR is
expressed in the stele of the root
(Helariutta et al., 2000
), but
SHR protein is found in both the stele and the surrounding endodermis, which
is missing in shr mutants. Further studies using transgenic
misexpression confirmed that movement of SHR from the root stele to endodermis
is required for endodermis development
(Nakajima et al., 2001
). RNA
of the plant-specific transcription factor LFY is expressed in all three
layers of young floral primordia, which are mis-specified as shoots in strong
lfy mutants (Weigel et al.,
1992
). Surprisingly, LFY RNA expression in the L1 of
developing flowers is sufficient to fully rescue the lfy-mutant
phenotype. In such transgenic plants, LFY protein, but not LFY RNA,
is detected in all layers of the rescued flowers, indicating that LFY protein
moves from the L1 into inner layers
(Sessions et al., 2000
). By
contrast, the transcription factor APETALA1 (AP1), which has similar in vivo
functions as LFY but is structurally unrelated, behaves largely
cell-autonomously (Sessions et al.,
2000
).
Although movement of transcription factors in Arabidopsis and other plants is by now well-established, there are still major gaps in understanding the underlying mechanisms. Here, we characterize the mode of LFY movement in Arabidopsis SAMs and floral primordia. Using functional LFY-GFP fusion proteins, we show that LFY moves more readily from the L1 into deeper cell layers than laterally into adjacent, clonally related cells. By contrast, a functional AP1-GFP fusion is unable to move from its source cells. Comparison of the dynamics of LFY-GFP fusion proteins with other GFP fusions suggests that this movement is driven by diffusion. Deletion experiments failed to identify a specific movement signal in LFY, which is compatible with the conclusion that LFY movement is non-targeted. The hypothesis of non-targeted movement is also supported by the finding of a correlation between cytoplasmic localization and the ability of these proteins to move to adjacent cells.
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MATERIALS AND METHODS |
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ML1::GFP:LFY was created by ligating the ML1 promoter
from pAS99 (Sessions et al.,
1999) to the 5' portion of GFP:LFY 5' (up to
the XbaI site in LFY) from pRTL2-sGFP:LFY, and the 3'
portion of LFY from pAS116 into binary vector pMX202. In this fusion,
there is an additional serine inserted between GFP and LFY.
35S::GFP:LFY was created by ligating the same GFP:LFY 5'
and LFY 3' fragments into binary vector pCHF3, which contains a
CaMV 35S promoter (Fankhauser et
al., 1999
).
ML1::LFY:GFP was created by ligating the 5' portion of
ML1::LFY 5' up to the HindIII site from pAS104
(Sessions et al., 2000) and
the 3' portion of LFY:GFP from pBS-ML1::LFY-link-sGFP into the
binary vector pJIHOON212 (J. H. Ahn, personal communication).
35S::LFY:GFP was created by ligating the 5' portion of
LFY cDNA from pAS107 (up to the HindIII site) with the
3' portion of LFY:GFP into pCHF3.
lfy mutant alleles
Four lfy alleles were included in this study: lfy-2,
lfy-3, lfy-9 and lfy-20
(Weigel et al., 1992).
GFP fusions of these mutant versions were generated in the same way
as the ML1::GLFY fusion.
LFY truncations
Truncations of the LFY coding sequence were created in the context of
pBS-LFY, which includes both the full-length cDNA and 300 bp of the
LFY 3'UTR. LFY1 was generated by
opening, filling-in and religating the BamHI site overlapping the
start codon and the XbaI site at position 379, which results in an
in-frame deletion of amino acids 4 to 127. LFY
2 was created by
opening, filling-in and religating the XbaI site at position 379 and
the StyI site at position 860, which results in an in-frame deletion
of amino acids 128 to 287. LFY
3 was created by opening,
filling-in and religating the StyI site at position 860 and the
HindIII site at position 974, which leads to a frame shift such that
amino acids 289 to 424 are replaced with the sequence SFKCSQKSV. Fusions of
the GFP:LFY truncations to the ML1 promoter were created by
combining the promoter fragment from pAS99, the 5' fragment of
GFP:LFY from pRTL2-sGFP:LFY and the respective LFY
truncations in pMX202.
AP1-GFP fusions
For AP1:GFP, restriction sites were added to the AP1 cDNA
sequence by PCR amplification, using pAM571 as a template (M. Yanofsky,
personal communication), which resulted in an EcoRI and a
BamHI site in front of the 5' UTR, and a PstI site at
the 3' end, replacing the stop codon. GFP coding sequence was
amplified from pCAMBIA1302, replacing the start codon with a PstI
site and adding an XbaI site to the 3'UTR. For
GFP:AP1, the start codon of AP1 was replaced with a
PstI site, and an XbaI site was added to the 3'UTR. An
EcoRI site was added to the 5' end of the GFP coding
region and a PstI site replaced the stop codon. In both fusions, the
PstI site also created an alanine linker of 2-3 amino acids.
ML1::AP1:GFP and ML1::GFP:AP1 were created by ligating the
ML1 promoter from pAS99 to the AP1 and GFP
fragments in the background of pMX202. AP1::AP1:GFP and
AP1::GFP:AP1 were created in the same way as the ML1
versions, but using the AP1 promoter from pAM571.
Other ML1 constructs
ML1::2xGFP was created by ligating the ML1
promoter to TEV5':2xsGFP from
pRTL2-2xsGFP (Crawford and Zambryski,
2000) and inserting into pMX202. The
NLS-2xsGFP fragment from pRTL2-NLS:2xsGFP
(Crawford and Zambryski, 2000
)
was used to generate ML1::NLS:2xGFP. ML1::TVCVMP:GFP
was generated by ligating the ML1 promoter to coding sequences for a
Turnip Vein Clearing Virus Movement Protein (TVCVMP):GFP fusion into
pMX202.
Ectopic expression in the center of shoot and flower meristems
A 833 bp BamHI/HindIII fragment from the 3' end of
the second AG intron (Busch et
al., 1999) was used to drive expression in the AG domain.
This enhancer fragment, from pMX141, carries a point mutation (from CCTTATTTGG
to AATTATTTGG) that results in ectopic activity in the
inflorescence meristem in a lfy-independent manner
(Hong et al., 2003
). The
enhancer was placed upstream of a -46 bp cauliflower mosaic virus 35S
minimal promoter in pMX202. AG intron*::2xGFP,
AG intron*::NLS:2xGFP, AG
intron*::LFY and AG intron*::GLFY were
created by inserting the respective coding sequence fragments into this
cassette.
LFY rescue constructs
The LFY and GLFY rescue constructs were generated by
expressing the LFY cDNA and GLFY under the control of the
2.3 kb LFY promoter
(Blázquez et al.,
1997).
Plant material
Plants were grown in long days (16 hour light / 8 hour darkness) under
120 µE m-2 seconds-1 light provided by a 3:1
mixture of cool-white and GroLux (Osram Sylvania) fluorescent bulbs, at
21°C. lfy-12 and ap1-15 are strong alleles in
the Columbia background (Huala and Sussex,
1992
; Ng and Yanofsky,
2001
). Plant transformations were carried out using the floral dip
method (Weigel and Glazebrook,
2002
). For each transgene, 40-70 T1 lines were initially analyzed,
and at least three plants each from three independent lines were used in
further characterization and imaging. Transgenic seedlings were selected on MS
agar plates containing 50 µg/ml kanamycin, then transplanted to soil. For
all transgenes presented in this work, multiple samples from both agar- and
soil-grown plants of at least three generations were examined, always with
very similar results. In all cases where a mutant allele was involved,
transgenic lines with both wild-type and mutant backgrounds have been
examined. We never observed an effect of the endogenous allele on the GFP
signal.
In situ hybridization
In situ hybridization was performed as described
(Sessions et al., 2000;
Weigel and Glazebrook, 2002
).
Digoxigenin-labeled antisense RNA probe for TVCVMP:GFP was generated by
digesting pBSTVCVMP:GFP with XhoI, then transcribing it using T3
polymerase,
Immunoblot analysis
Crude protein extract was obtained from 2-week-old seedlings and separated
on 4-12% gradient gels (NuPAGE, Invitrogen) with Benchmark Protein Marker
(Invitrogen). Samples were transferred to a PVDF membrane by electroblotting,
and incubated with rabbit anti-GFP primary antibody (1:1000 dilution,
Molecular Probes). An HRP-conjugated goat anti-rabbit secondary antibody
(1:5000 dilution, BioRad) was used for signal detection with SuperSignal
Chemiluminescent Substrate (Pierce).
Microscopy
GFP fluorescence images
For ML1 transgenic plants, emerging leaves and apices were
dissected from 10- to 12-day-old seedlings grown on MS agar plates. For AG
intron* transgenic plants, primary inflorescence apices were
dissected from 4-week-old plants for image analysis. Confocal images were
collected using a 40x or 63x oil-immersion lens on a Leica SPII
spectral confocal laser scanning microscope. GFP fluorescence was excited with
a 488 nm Argon laser, and images were collected in the 500-550 nm range. In
some cases, images from the transmissible light channel were collected
simultaneously. For ML1 transgenic plants, both vegetative and
inflorescence apices were examined. In each figure, all panels were collected
during the same microscopy session from plants grown under exactly the same
conditions.
Light microscopic images
Pictures of flowers were taken with a Polaroid DMC digital camera mounted
on an Olympus SZH10 stereomicroscope. Images of in situ hybridization were
taken with a SPOT digital camera mounted on a Nikon compound microscope.
Quantification of the subcellular distribution of GFP-LFY
fusions
Confocal image series of epidermal cells in emerging leaves were collected
from transgenic plants carrying ML1::GLFY, ML1::GFP:LFY, and ML1::LFY:GFP
using a 63x objective, with 1 µm steps. Separate masks were generated
for the nuclei and the cytoplasm using the image processing software Khoros
(www.khoral.com).
Total signal intensity in each part was calculated for every section and
summed within the same image series. Data from approximately thirty cells were
averaged for each transgene.
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RESULTS |
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To confirm that the GFP signal was indeed from dimerized GFP instead of a breakdown product resulting in monomeric GFP, whole-cell protein extracts were prepared from transgenic seedlings after imaging and analyzed by western blot with an anti-GFP antibody. For NLS:2xGFP, a major band at about 57 kDa was observed (predicted size 58 kDa), indicating that it was stable (Fig. 4A). Similarly, 2xGFP migrated at about 50 kDa (predicted size 54 kDa), and only a small amount of degradation product was observed for 2xGFP.
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Movement of LFY-GFP fusions from the L1 layer
To avoid artifacts caused by the addition of GFP to a specific domain of
the LFY protein, we generated three different LFYGFP fusions: GFP:LFY, an
N-terminal fusion; GLFY, with an insertion of GFP at amino acid 31; and
LFY:GFP, a C-terminal fusion. We used these fusions, which increase the size
of LFY by about half, from 47 to 74 kDa, to further characterize the movement
of LFY, which has been previously detected using anti-LFY antibodies
(Sessions et al., 2000). The
fusions were introduced into lfy-12/+ plants under the control of the
ML1 promoter. To test independently for functionality, the N- and
C-terminally tagged versions were also expressed under the control of the
constitutive CaMV 35S promoter. All five transgenes were able to
rescue the lfy-12 mutant phenotype, and to cause the typical
gain-of-function phenotypes associated with overexpression of LFY
(Weigel and Nilsson, 1995
),
which indicates that the three GFP fusions were fully functional.
Using the ML1 promoter lines, we examined the subcellular localization of the LFY-GFP fusions, as well as their movement from the L1 layer (Fig. 3). All three fusions were detected in both the nucleus and cytoplasm, which was best seen in leaf epidermal cells (GLFY shown in Fig. 3C), and all produced more cytoplasmic signal than NLS:2xGFP (Fig. 1F). In leaf epidermal cells, a punctate signal that appeared along the cell wall was observed with all three fusions (GLFY shown in Fig. 3C), which suggests a possible association with plasmodesmata pit fields. GLFY and GFP:LFY moved three to four cell layers into the L2 and L3 in both the vegetative and inflorescence apices, forming a gradient with the highest concentration in the L1 (Fig. 3A,B). Both GLFY and GFP:LFY were restricted to the epidermal layer in maturing leaves (data not shown). LFY:GFP moved further, approximately 10 cell layers in apices (Fig. 3D). In leaves, its distribution in the epidermis was similar to that of GLFY and GFP:LFY, but it could also be detected in the underlying mesophyll cells (data not shown). Overall, LFY:GFP appeared more cytoplasmic than GLFY and GFP:LFY. This observation was confirmed by quantifying the total signal intensity in the nucleus and cytoplasm of the epidermal cells of emerging leaves from all three fusions (see Materials and Methods). For GLFY and GFP:LFY, the nuclear to cytoplasmic signal ratios were very similar, 1:2.3 and 1:2.5, respectively. By contrast, the nuclear to cytoplasmic ratio of LFY:GFP was 1:5.4, a twofold increase compared with GLFY and GFP:LFY. Western blots probed with an anti-GFP antibody demonstrated that there was little degradation of the fusion proteins, indicating that the in vivo fluorescence signal came from the intact fusion proteins (Fig. 4C). Taken together, the behavior of LFY-GFP fusions is intermediate between that of 2xGFP and NLS:2xGPF, both with respect to their cytoplasmic localization and their ability to move from the L1 layer.
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We expressed 2xGFP, NLS:2xGFP and GLFY under the control of the CaMV 35S minimal promoter fused to the mutated AG enhancer (AG intron*). Except for the subcellular localization, the expression patterns of NLS:2xGFP (Fig. 5A) and 2xGFP (Fig. 5B) were indistinguishable, with fluorescent signal in the inflorescence meristem and central domain of young floral primordia. The signal in both the shoot and floral meristems had discrete boundaries, indicating that GFP neither moved from the inflorescence meristem into emerging floral primordia, nor moved from the center of stage 3 flowers to the periphery (Fig. 5D,E). However, a gradient of 2xGFP could be seen extending into deeper cell layers in L3 in stage 3 flowers (Fig. 5E), which is consistent with our earlier observation. Thus, compared with movement from the L1 to internal layers, lateral movement of 2xGFP and NLS:2xGFP within tissue layers is much more limited, or possibly even absent. The much reduced movement of 2xGFP and NLS:2xGFP within L1 and L2, compared with movement between layers, suggests that the plasmodesmata SEL within these two layers is lower than that between layers.
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We used a functional assay to test this assumption. For this, we took advantage of the fact that the mutated AG enhancer is active in lfy mutants. When we expressed LFY and GLFY under the control of this enhancer in lfy-12 plants, the same fraction of transgenic lines showed rescue in flowers (4 out of 12 lines for LFY and 4 out of 11 lines for GLFY; no significant difference using Fisher's exact test), suggesting that GLFY has similar activity to LFY. However, the rescued flowers differed in phenotype. Both GLFY and LFY rescued the development of the two inner whorls, which contain stamens and carpels, and in which the AG enhancer is active (Fig. 6), but only LFY was able to rescue petal development. All four of the lfy-12; AG intron*::LFY lines that showed phenotypic rescue produced at least some flowers with petals, and many flowers had the normal complement of four petals. By contrast, none of the four lfy-12; AG intron*::GLFY lines produced flowers with petals. As GLFY appears to be as active as LFY in the inner two whorls where it is produced, the difference in their activity in the outer two whorls is consistent with the conclusion that LFY can move more extensively than GLFY.
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Movement of mutant LFY proteins
To further test whether LFY movement is regulated, we investigated whether
deleting parts of the LFY protein abolishes intercellular movement. Three
large, non-overlapping deletions were made in the LFY coding sequence, each
removing approximately one third of the protein
(Fig. 7A). All three were
linked to GFP at the N terminus and expressed under the ML1 promoter.
Although they differed in the extent with which they moved from L1 to inner
layers, all three deletion variants were still able to move from L1 into the
inner layers in both vegetative and inflorescence apices. GFP:LFY1,
with an N-terminal deletion, behaved very similar to GFP:LFY. It was mostly
located in the nucleus, and formed a gradient of four to five cell layers into
the L2 and L3 (Fig. 7B). GFP:LFY
2, with a central deletion, was expressed at lower levels and
was largely cytoplasmic, presumably because of the deletion of the NLS. GFP
signal could be clearly detected for at least three cell layers into the L2
and L3 (Fig. 7C), but its low
expression levels may have been limiting our ability to determine its actual
range of movement. GFP:LFY
3, with a C-terminal deletion, showed the
least degree of movement, moving only one to two cell layers from the L1
(Fig. 7D). However, most of the
GFP signal was found in large aggregates, sometimes associated with the cell
membrane when imaged in the leaf epidermis (data not shown), suggesting that
GFP:LFY
3 is improperly folded and localizes to a specific subcellular
compartment, which may affect its movement. Furthermore, all three truncated
versions were able to enter mesophyll cells from the epidermis in maturing
leaves (data not shown).
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DISCUSSION |
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Mode of LFY movement
Several lines of evidence are compatible with the view that LFY movement is
non-targeted. First, GFP-LFY fusions produced in the L1 formed limited
gradients extending into deeper layers. Their movement range was between that
of 2xGFP and NLS:2xGFP, which move in a non-targeted fashion
(Crawford and Zambryski, 1999;
Crawford and Zambryski, 2000
;
Kim et al., 2002
). This is in
contrast to the nearly uniform distribution in the shoot apex (reflecting an
active mechanism of cell-to-cell transport) that is observed when a viral
movement protein fusion is expressed in the L1. Second, size affects LFY
movement, as native LFY was more effective in rescuing lfy defects in
adjacent cells in the same tissue layer than the larger GLFY fusion. The
effect of size is one of the prominent characteristics of non-targeted
movement (Zambryski and Crawford,
2000
). Third, although differing in stability and sub-cellular
localization, all three GFP-LFY truncations and fusions of four mutant
lfy alleles were able to move from the L1 into interior cell layers.
Although one cannot exclude the possibility that LFY has several redundant
movement signals, the simpler explanation is that the LFY protein sequence
does not contain a specific movement or export signal.
It has been suggested that non-targeted movement, like targeted movement,
occurs through plasmodesmata. The potential localization of foci of GFP-LFY
fusion proteins along the cell wall in the leaf epidermis supports this
hypothesis. This also suggests that the size exclusion limit of the secondary
plasmodesmata connecting tissue layers in the Arabidopsis apex is
greater than 74 kDa (the size of the LFY-GFP fusions), which is consistent
with previous estimates for nascent leaves
(Zambryski and Crawford,
2000).
Movement within and between tissue layers
An important new finding is that GFP variants, as well as GLFY, move more
easily in the apical-basal direction than laterally. Using in vivo function as
a criterion, we found that the GLFY fusion was less efficient than the native
LFY in moving from the center of floral primordia to the periphery. This
functional difference is most likely a result of their size difference,
because both proteins are fully functional in the cells where they are
produced. Similar to previous studies that have demonstrated that the inner
central zone (L3) does not allow fluorescent tracer uploading from the
vascular tissue (Gisel et al.,
1999; Gisel et al.,
2002
), we observed much more limited lateral movement within the
L3. Thus, intercellular movement needs to be considered in the context of the
specific location and developmental stage within the plant.
That lateral movement is less easily achieved than apical-basal movement
may also explain the fact that LFY does not move out of floral primordia into
the inflorescence meristem in wild type
(Parcy et al., 1998;
Sessions et al., 2000
), as
lateral movement would be required for efficient protein exchange between the
two tissues. The inflorescence meristem may even form a symplasmic domain that
is insulated from emerging floral primordia
(Rinne and van der Schoot,
1998
), thus restricting movement of all macromolecules.
Alternatively, there may be selective gating, such that movement of only
certain macromolecules from floral primordia into the inflorescence meristem
(and vice versa) is permitted. A similar mechanism may also be responsible for
maintaining discrete whorl boundaries within the flower. In this context, it
is noteworthy that floral homeotic proteins that are expressed in distinct
whorls of the developing Arabidopsis flower, such as AP1 and AP3, do
not move, whereas LFY, which is expressed throughout the flower, does
(Jenik and Irish, 2001
;
Sessions et al., 2000
).
Movement and subcellular localization
LFY and LFY-GFP fusions can move into the inner tissue layers from the L1.
By contrast, AP1:GFP does not move between tissue layers. This cannot be
simply due to size, because an N-terminal fusion, GFP:AP1, could move well.
Furthermore, GFP:AP1 (55 kDa) is smaller than either NLS:2xGFP (57 kDa)
or LFY-GFP fusions (74 kDa). Therefore, if LFY is moving by diffusion, AP1 and
AP1:GFP must be actively retained in the cells where they are expressed. One
way to achieve the retention may be by subcellular localization, such as
nuclear or ER localization. From this study, we have found that there is a
good correlation between nuclear localization and movement: 2xGFP, which
is highly cytoplasmic, can move a considerable distance from the L1, and the
same is true for the predominantly cytoplasmic GFP:AP1 fusion. AP1:GFP, which
appeared to be exclusively nuclear, did not move. Between these two extremes,
NLS:2xGFP showed little cytoplasmic localization and moved only one cell
layer. The GFP-LFY fusions all showed more cytoplasmic localization than
NLS:2xGFP, and all moved farther than NLS:2xGFP but less than
2xGFP. Among the three GFP-LFY fusions, LFY:GFP had the most cytoplasmic
localization and moved the farthest.
Another possible mechanism for retaining a protein could be through the
formation of large protein complexes with more exclusive subcellular
localization, or simply with sizes above the SEL of plasmodesmata. This may
contribute to the retention of MADS domain proteins such as AP1, as several of
them, including AP1, are known to form heteromultimers in the absence of DNA
(Egea-Cortines et al., 1999;
Honma and Goto, 2001
). In
addition, it has been shown that AP3, a MADS-domain transcription factor that
does not move between tissue layers, needs to heterodimerize with another
MADS-domain protein, PISTILLATA (PI), in order to localize to the nucleus
(McGonigle et al., 1996
). It
is possible that the GFP:AP1 fusion disrupts such interactions, thereby
interfering with biological activity and nuclear localization, as well as with
retention in the cells where it is produced.
In this context, it is noteworthy that SHR is found in both the nucleus and
cytoplasm of the stele, where it is produced. From there, SHR moves exactly
one cell diameter, into the adjacent endodermis, where it is located entirely
in the nucleus (Nakajima et al.,
2001). This observation is consistent with a model in which SHR
gets trapped in the nuclei of the endodermis through interaction with a
partner that causes translocation to the nucleus, similar to the AP3/PI
interaction (McGonigle et al.,
1996
).
Mechanisms of movement
Our results are compatible with the view that LFY movement is driven by
diffusion. However, it remains unclear whether the same conclusion can be
drawn regarding the intercellular movement of other transcription factors.
Another well-studied example of a trafficking transcription factor is KN1 of
maize (Kim et al., 2002;
Kragler et al., 2000
;
Lucas et al., 1995
). As with
LFY, KN1-GFP fusions are detected in the nucleus and cytoplasm, and in a
punctate pattern associated with the cell wall
(Kim et al., 2002
). In
contrast to LFY, for which various deletions did not prevent movement, a
simple mutation in the homeodomain and the potential NLS of KN1 abolished its
movement. Furthermore, experiments with tobacco have indicated the presence of
a cellular component that is limiting for KN1 movement, and have suggested
that the mode of KN1 trafficking may be related to the targeted movement of
viral movement proteins (Kragler et al.,
2000
). However, other results are consistent with KN1 moving in a
non-targeted fashion. In bombardment assays, KN1-GFP moved considerably less
well than a fusion of GFP to the movement protein of TVCV
(Kim et al., 2002
).
Furthermore, movement of KN1 expressed from the SCR promoter in the
shoot apex of transgenic Arabidopsis plants was rather limited
(Kim et al., 2002
), similar to
that of NLS:2xGFP or GFP:LFY expressed from the ML1 promoter
(this work). Further studies in a nonheterologous system will be required to
clarify the mechanisms behind KN1 movement. A complex relationship between
targeted and non-targeted movement is also indicated by a recent study, in
which a dominant-negative form of the tobacco NON-CELL-AUTONOMOUS PATHWAY
PROTEIN 1 (NtNCAPP1) was overexpressed. In such transgenic plants, trafficking
of viral movement protein, but not of KN1, was affected
(Lee et al., 2003
).
Interestingly, tobacco LFY protein appears more uniform in such transgenic
plants, which also have phenotypes reminiscent of LFY overexpressing plants.
The causal relationship between these observations needs further
investigation, but it will be interesting to combine the dominant-negative
NtCAPP1 overexpressing plants with the tools presented here.
It is also worth noting that the determinants underlying transcription
factor movement may be species-dependent. Like LFY, its Antirrhinum
ortholog FLORICAULA (FLO) has non-cell-autonomous effects in mosaic studies
(Carpenter and Coen, 1995). In
contrast to LFY, the extent to which FLO can rescue mutant flowers varies
depending on the layer in which FLO is expressed
(Hantke et al., 1995
;
Sessions et al., 2000
). As FLO
protein has not been examined in these mosaics, it is unknown whether the
differential rescue ability is caused by differences in FLO movement, or is
only a result of downstream effects, such as the documented abnormalities in
target gene expression in the mosaics
(Hantke et al., 1995
). Another
example of interspecific differences is provided by DEF of
Antirrhinum, which was found to move from the L2 to the L1 in a
stage- and organ-dependent manner (Perbal
et al., 1996
). However, the DEF ortholog AP3 of
Arabidopsis does not move between layers
(Jenik and Irish, 2001
), which
indicates that subtle differences in sequence, or interspecific differences in
the translocation machinery, affect transcription factor movement, the latter
being consistent with the interspecific differences that have been reported
for GFP movement (Crawford and Zambryski,
2001
). These observations highlight that care must be taken when
extrapolating from one transcription factor assayed in a single species or
single tissue.
In conclusion, we have presented evidence that the transcription factor LFY moves in a non-targeted fashion. We are proposing the testable hypothesis that movement is a default mechanism for many proteins in the Arabidopsis shoot apex, unless they are either efficiently targeted to specific subcellular locations or retained through formation of protein complexes. More case studies are needed to determine whether our results can indeed be generalized to include other proteins, other tissues and other species.
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ACKNOWLEDGMENTS |
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