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Differential Expression Control and Polarized Distribution
of Plasma Membrane-Resident SYP1 SNAREs
in Arabidopsis thaliana
Kazuhiko Enami1, Mie Ichikawa1, Tomohiro Uemura2, Natsumaro Kutsuna3,4, Seiichiro Hasezawa3,4,
Tsuyoshi Nakagawa5, Akihiko Nakano2,6 and Masa H. Sato1,*
1Laboratory
of Cellular Dynamics, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5, Shimogamonakaragi-cho, Sakyo-ku, Kyoto, 606-8522 Japan
2Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033
Japan
3Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, 277-8562 Japan
4Institute for Bioinformatics Research and Development (BIRD), Japan Science and Technology Agency (JST), Japan
5Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, Matsue, 690-8504
Japan
6Molecular Membrane Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama, 351-0198 Japan
Membrane trafficking to the plasma membrane (PM) is a
highly organized process which enables plant cells to build
up their bodies. SNARE (soluble N-ethylmaleimidesensitive factor attachment protein receptor) genes, which
encode the proteins involved in membrane trafficking, are
much more abundant in the Arabidopsis genome than in
that of any other eukaryote. We have previously shown
that a large number of SNARE molecules in the Arabidopsis
cell are localized predominantly on the PM. In the present
study, in order to elucidate the physiological function of
each PM-localized SNARE, we analyzed the spatiotemporal
expression profiling of nine SYP1s that are resident in the
PM of Arabidopsis, and used the information thus acquired
to generate transgenic Arabidopsis plants expressing
green fluorescent protein-fused Qa-SNAREs under control
of their authentic promoters. Among the nine SYP1s, only
SYP132 is expressed ubiquitously in all tissues throughout
plant development. The expression patterns of the other
SYP1s, in contrast, are tissue specific, and all different from
one another. A particularly noteworthy example is SYP123,
which is predominantly expressed in root hair cells during
root development, and shows a focal accumulation pattern
at the tip region of root hairs. These results suggest that
SYP132 is involved in constitutive membrane trafficking
to the PM throughout plant development, while the other
SYP1s are involved in membrane trafficking events such as
root formation or tip growth of root hair, with some
redundancy.
Keywords: Arabidopsis • Membrane traffic • Plasma membrane
• SNARE • Tip growth
Abbreviations: DIC, differential interference contrast; FTFLP,
fluorescent tagging of full-length proteins; GFP, green
fluorescent protein; mRFP, monomeric red fluorescent
protein; ORF, open reading frame; PM, plasma membrane;
SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TT-PCR, triple-template PCR.
Introduction
SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) molecules play an essential role in the
membrane fusion event that occurs at the final step of membrane trafficking. SNAREs are one of the most conservative
molecules in eukaryotes, and constitute a large superfamily
found in all eukaryotes. They are classified as either Q-SNARE
(Qa, Qb and Qc) or R-SNARE according to their conserved
residues within the SNARE motif (Fasshauer et al. 1998, Bock
et al. 2001). During the membrane fusion process, a specific
set of three distinct Q-SNARE molecules and one R-SNARE
molecule form a transSNARE complex on the membrane
of each organelle. In order to complete the process of
*Corresponding author: E-mail, [email protected]; Fax, +81-75-703-5448.
Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197, available online at www.pcp.oxfordjournals.org
© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197 © The Author 2008.
Spatiotemporal expression control of PM SNAREs
membrane fusion on a particular organelle, the appropriate
set of SNARE molecules is required in the transport pathway
to that organelle.
The family of SNAREs found in Arabidopsis is encoded by
>60 genes (Yoshizawa et al. 2006, Sanderfoot 2007; reviewed
in Lipka et al. 2007); this is a greater number of SNARE genes
than that found in any yeast or animal. We have previously
determined the subcellular localization of almost all SNARE
molecules in Arabidopsis by transient expression analysis of
green fluoresent protein (GFP)-fused SNARE molecules, and
found that many SNARE molecules are localized on the
plasma membrane (PM). Among the 17 Qa-SNARE group
proteins, a total of nine (SYP111, SYP112, SYP121, SYP122,
SYP123, SYP124, SYP125, SYP131 and SYP132) are localized
on the PM (Uemura et al. 2004).
Functional analyses have already been performed for
some of these PM-Qa SNAREs. The KNOLLE/SYP111 protein,
for example, is specifically expressed during mitosis and
relocated to the forming cell plate, thereby functioning in
cytokinesis (Lukowitz et al. 1996, Lauber et al. 1997). As for
SYP121, an ortholog of it was first isolated from tobacco as
an ABA response-related molecule, NtSyr1 (Leyman et al.
1999). The expression of the cytosolic domain of NtSyr1 or
SYP121 causes inhibition of accurate secretion of secGFP
and changes in the mobility of GFP–KAT1 on the PM, suggesting that SYP121 is involved in secretion and is required
for cellular growth and ion homeostasis (Geelen et al. 2002,
Sutter et al. 2006, Tyrrell et al. 2007). SYP121 was also identified as the molecule that is involved in non-host resistance
against powdery mildew; a mutation in AtPEN1/SYP121 and
its barley ortholog HvROR2 allowed enhanced penetration
of the powdery mildew fungus Blumeria graminis f. sp. hordei
(Collins et al. 2003). SYP122, a paralog of SYP121, is phosphorylated in response to elicitor flagellin (Nuhse et al. 2003).
SYP121 and SYP122 are induced by the stimulation of fungal,
bacterial and/or viral pathogens; at the time of non-host
fungal infection, these molecules accumulate at the infection site to form papilla (Assaad et al. 2004). A syp121 syp122
double mutant exhibited a dwarfed and necrotic phenotype,
suggesting that these two PM SNARE molecules have redundant functions not only in plant immunity but also in general secretion events (Assaad et al. 2004, Zhang et al. 2007).
Recent progress in PM Qa-SNARE research has revealed that
SYP132 orthologs also play roles in bacterial defense
and symbiosome definition in Nicotiana benthamiana and
Medicago truncatula, respectively (Catalano et al. 2007, Kalde
et al. 2007). These data suggest that PM-resident Qa-SNARE
molecules function in different membrane trafficking pathways leading to the particular domains of the PM surface,
with some redundancy so that they can transport various
functional molecules in a polarized or non-polarized
manner.
Although some PM Qa-SNARE molecules have been well
characterized, no PM Qa-SNARE involved in polarized membrane trafficking to the PM, such as that involved in tip
growth, has been identified so far. In order to identify some
of the Qa-SNARE molecules involved in these processes, we
generated transgenic plants expressing GFP-fused PM
Qa-SYP1s under control of their own promoters, using the
fluorescent tagging of full-length proteins (FTFLP) technique
(Tian et al. 2004). This technique offers us a strong advantage in analyzing the expression patterns of the target genes
and the subcellular distributions of their protein products
simultaneously. Here we report that one SYP12 molecule,
SYP123, shows strong focal accumulation on the tip region
of the root hair, suggesting that it might be involved in the
polarized elongation process of root hairs. In contrast,
SYP132 is ubiquitously expressed in all tissues so far examined, and is localized uniformly on the PM. We also observed
complicated tissue-specific expression patterns for each
GFP–SYP1 SNARE, indicating that the physiological roles of
PM Qa-SNAREs are probably independent of each other,
although with partial redundancy.
Results
Expression profiles of SYP1 genes in various
Arabidopsis organs
Based on a phylogenic analysis of the SNAREs found in green
plants, Sanderfoot (2007) categorized the PM-resident SYP1
clade into two large groups, SYP12 and SYP13. The SYP12
group is further divided into four distinct subgroups. Up to
five subgroups of Qa-SNAREs may exist on the PM, namely
SYP13 (SYP131 and SYP132), PEN1/ROR2 (SYP121 and
SYP122), SYP124 (SYP123, SYP124 and SYP125), SYP11/
KNOLLE (SYP111/KNOLLE) and SYP112. Using the Genevestigator V3 microarray database (Zimmermann et al. 2004,
Hruz et al. 2008), we analyzed the tissue-specific gene expression patterns of all SYP1 group SNAREs. As shown in Supplementary Fig. S1, SYP111 is highly expressed in organs
containing dividing cells, such as root tip, silique and shoot
apex, whereas its functional paralog, SYP112, is expressed to
a lesser degree throughout all tissues so far examined. The
PEN1/ROR2 subgroup Qa-SNAREs, SYP121 and SYP122,
show similar expression patterns, but the expression of
SYP121 is slightly higher than that of SYP122 in the lateral
root cap. SYP123, one of the SYP124 subgroup SNAREs, is
predominantly expressed in particular root tissues including
the elongation zone, root hair zone and the lateral root. In
contrast, the other SYP124 subgroup SNAREs, SYP124 and
SYP125, are expressed only in pollen. One SYP13 subgroup
SNARE, SYP131, is also specifically expressed in pollen,
whereas SYP132, another member of this subgroup, shows
a ubiquitous expression throughout all tissues. Thus, these
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PM Qa-SNAREs are expressed differently among Arabidopsis
organs, suggesting that these SNAREs might have distinct
functions in plant development.
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SYP1 genes are expressed differently during different
periods of root development
We generated transgenic Arabidopsis plants expressing GFPfused PM-resident SYP1 SNAREs under control of their
native promoters, and simultaneously observed the expression patterns and subcellular localization with confocal laser
microscopy.
Among the PM-resident Qa-SNARE molecules, five
(SYP111, SYP121, SYP122, SYP123 and SYP132) were
expressed in certain root tissues of 5-day-old seedlings. The
GFP fluorescence of SYP111/KNOLLE was detected exclusively in dividing cells in the root tip region (Fig. 1A). The
majority of expressed SYP111 proteins were predominantly
localized to the forming cell plates during cytokinesis (arrows
in Fig. 1C), although faint fluorescent signals were also
observed on the intracellular punctate structures around
the cell plate in the same cells (arrowheads in Fig. 1C). These
punctate GFP signals partially overlapped with the fluorescence of an endocytosis marker, FM4-64 (Fig. 1E), indicating
that GFP–SYP111 was localized to various punctate organelles including the Golgi and trans-Golgi network, as well as
to the endosomes (Reichardt et al. 2007). No intracellular
signals were detected in non-dividing cells anywhere in the
root tissues including the root tip. The localization pattern
of GFP–SYP111 observed here was completely consistent
with the localization pattern of authentic SYP111 that was
shown in an indirect immunofluorescent experiment (Volker
et al. 2001). These results imply that our own promoter
system revealed the precise subcellular localizations and
tissue-specific expression of SNARE molecules throughout
the plant body.
In contrast to SYP111 with its characteristic restricted
expression pattern, SYP132 was expressed ubiquitously in
almost all cells in the root tissues, including the root apical
meristem, epidermis, cortex, endodermis, stele and root hair
cell (Fig. 1F, H, I). Although in non-dividing cells the fluorescence of GFP–SYP132 was only detected on the PM, in dividing
cells its fluorescence was also observed in cytosolic punctate
structures in addition to the cell plates (asterisks in Fig. 1F).
It has been reported elsewhere that PM-localized Qa-SNARE
molecules can be detected in FM4-64-positive endosomes
(Uemura et al. 2004); accordingly, we believe that the GFP–
SYP132-containing punctate structures we observed were
the endosomes.
SYP121 was expressed predominantly in the epidermal
cells and lateral root cap cells in the root tip region (Fig. 2A).
The expression in lateral root cap cells observed with
GFP fluorescence is consistent with the microarray data
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Fig. 1 Expression patterns of GFP–SYP111 and GFP–SYP132 in root.
(A and B) A projection image of confocal sections of the fluorescence
of GFP–SYP111 (A), and the corresponding DIC image (B) in a 5-dayold seedling of a transgenic plant expressing GFP–SYP111 under
control of the SYP111 promoter. (C–E) Co-localization of GFP–SYP111
with FM4-64 in the dividing cell. Higher magnification images of the
confocal sections of dividing cells expressing GFP–SYP111 were taken
1 h after the FM4-64 treatment. A merged image of GFP (C) and FM464 (D) is depicted in (E). Two fluorescence signals were overlapped
at the cell plates (arrows) as well as at the subcellular punctate
structures (arrowheads). (F–I) Confocal section images of GFP–
SYP132 fluorescence in the root (F, H, I) and the corresponding DIC
images (G). The root tip region (F, G) and mature root region (H, I) of
the 5-day-old seedlings were observed. Asterisks in F indicate dividing
cells. ep, epidermis; co, cortex; en, endodermis; st, stele. Bars = 20 µm
(A, F, H, I) and 10 µm (C).
Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197 © The Author 2008.
Spatiotemporal expression control of PM SNAREs
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Fig. 2 SYP121 and SYP122 are differentially expressed in root tissues. The confocal images of GFP fluorescence (A, C, E, G, I, K) and the DIC
(B, D, F, H, J, L) images of root sections of 5-day-old transgenic plants expressing either GFP–SYP121 (A, B, E, F, I, J) or GFP–SYP122 (C, D, G, H, K, L);
root tip (A–D), root hair elongation zone (E–H), mature root (I–L). Projection images are shown in E and G. Asterisks indicate the point where
root hairs start to bulge. lrc, lateral root cap; ep, epidermis; co, cortex; en, endodermis; st, stele. Bars = 20 µm.
(Supplementary Fig. S1). The epidermal expression of
SYP121 was observed in the whole root, but the intensity of
the fluorescence decreased during root development. Particularly in root hair cells, GFP fluorescence was weaker in
the differentiation zone, where root hairs begin to develop
(Fig. 2E and Supplementary Fig. S2A–C). Finally, weak GFP
signals were constantly observed in all types of cells in various tissues, including the cortex, endodermis and stele, in
addition to the epidermal cells in the mature root region
(Fig. 2I). Notably, GFP–SYP121 formed patch-like structures
which were scattered on the PM in addition to its normal
PM localization (Fig. 2E). Longitudinal imaging of GFP–
SYP121 in the root showed that these patch-like structures
protruded slightly into the cytosolic region from the PM
(Supplementary Fig. S2A–C). Time-lapse observation
revealed that several of these dot structures of GFP–SYP121
moved dynamically along the surface of PM (Supplementary Fig. S3, arrows), whereas predominant fluorescent dots
were less mobile on the PM (Supplementary Fig S3,
arrowheads).
The expression profile of GFP-tagged SYP122 during root
development was completely different from that of SYP121.
In the root tip region, no fluorescence was observed in any
cells examined (Fig. 2C). Instead, GFP fluorescence appeared
in the endodermal cells at the differentiation zone (Fig. 2G).
In the mature root region, a weak expression of GFP–SYP122
was observed in the cortex and epidermis, in addition to the
strong expression in the endodermal cells (Fig. 2K and Supplementary Fig. S2D–F). Thus the expression patterns and
intensities of these PM Qa-SNAREs varied according to cell
types in the root section, although SNAREs were uniformly
localized to the PM of all types of cells in root so far
examined.
Intriguingly, the expression pattern of GFP–SYP123 is
notably different from those of the other PM-resident QaSNARE molecules described above. No expression of GFP–
SYP123 was observed at the root tip region (data not shown),
but, after the root hair bulge rose at the differentiate region,
GFP fluorescence was specifically observed in the root hair
cells (Fig. 3D). The high-resolution expression analysis of
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Fig. 3 GFP–SYP123 is predominantly expressed in root hair cells and accumulates in the root hair tip region. Five-day-old seedlings expressing
GFP–SYP123 were observed and analyzed. Section images of a single root hair cell (A–C) and projection image of the root hair elongation region
(D). As root hairs elongate, a strong GFP signal is observed at the PM of the root hair region. Note that GFP fluorescence can be observed after
the root hair bulges emerged. The region surrounded by a dotted square in F was analyzed and constructed into a 3D image with ReantLight
software (E). Color represents normalized fluorescence intensity (10–220 arbitrary units) which correlates with GFP–SYP123 density, estimated
by 3D deconvolution to reduce the bias from point-spread function anisotropy (Sano et al. 2008). The intensity seen in the root hair (from sky-blue
to red) was higher than that in the base cell (blue) and peaked at the tip of the root hair (from green to red). Bars = 20 µm.
root tissues also revealed that SYP123 is enriched in root hair
cells (Brady et al. 2007). Although the fluorescence signals of
GFP–SYP123 continued to be visible in mature root hair
cells, their intensity was significantly reduced (data not
shown). No expression of GFP–SYP123 was observed in any
other tissues, not even in non-root hair cells. It should be
noted that once the root hair elongation began at the root
hair cells, GFP fluorescence accumulated predominantly in
the PM of the developing root hair region, especially at the
tip of the root hair (Fig. 3A–C). 3D image construction using
ReantLight software provided us with a clear view of the
strong accumulation of GFP–SYP123 at the tip region of
each root hair (Fig. 3D, E). This distribution pattern of
SYP123 in root hair cells is obviously different from that
of SYP132 (compare Fig. 3E with Fig. 1I or Supplementary
Fig. S4). These data suggest that the expression of SYP123 is
specific to root hair cells, and that it might be involved in
root hair elongation.
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Expression profiles of SYP1 genes in the aerial part of
Arabidopsis plants
Next, we observed the aerial part of each transgenic plant
expressing GFP-fused PM Qa-SNAREs. GFP–SYP111 was specifically expressed and localized to the developing cell plates
in the dividing cells during guard cell development, which
suggests that SYP111 might also be involved in the formation of guard cells (Supplementary Fig. S5A). Both SYP121
and SYP122 were uniformly localized to the PM of epidermal
cells in mature rosette leaves, even in the absence of any
fungal infection (Supplementary Fig. S5D, E). SYP132
showed a strong steady-state expression in all rosette leaf
cells throughout all stages of leaf development (Supplementary Fig. S5G). In the GFP–SYP123-expressing plant, on
the other hand, clear GFP fluorescence was only observed in
the epidermal cells of young rosette leaves (Supplementary
Fig. S5F). The intensity of GFP–SYP123 fluorescence attenuated gradually during leaf development, and only faint
Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197 © The Author 2008.
Spatiotemporal expression control of PM SNAREs
signals of this SNARE remained in the mature epidermal cells
of rosette leaves (data not shown).
SYP121, SYP123 and SYP132 were also expressed in
trichome cells of rosette leaves and the inflorescence stem.
The GFP fluorescence of these GFP–SNAREs was observed
uniformly on the PM of the cells (Supplementary
Fig. S5H–J). In the inflorescence stem, expression of the
SYP12 subgroup SNAREs (SYP121 and SYP123) was restricted
to the epidermal cell layer, whereas the fluorescence of GFP–
SYP132 was observed not only in epidermal cells but also in
mesophyll cells (Supplementary Fig. S6A–C). No GFP fluorescence on the PM was ever observed in guard cells or trichome
cells of wild-type Arabidopsis (Supplementary Fig. S7).
In flower tissues, the expression of GFP–SYP121, GFP–
SYP123 and GFP–SYP132 was detected in epidermal cells.
Strong expression of GFP–SYP123 was observed in epidermal cells around the top of the stigma, while none was
detected in the stigmatic papilla at flowering stage 11 (Supplementary Fig. S5K). In mature pollen grains, SYP132 was
expressed with clear PM localization (Fig. 4G), although
other SYP1s which were expressed in vegetative tissues were
not expressed in pollen grains (data not shown). In contrast,
SYP124, SYP125 and SYP131 were specifically expressed in
the pollen grains (Fig. 4A, C, E). Intriguingly, these SNAREs
did not show clear subcellular localization patterns on the
PM. Instead, faint GFP signals were observed throughout
the cytoplasm of pollen grains. We compared these GFP
signals with the fluorescence pattern of a vacuolar membrane marker, monomeric red fluorescent protein (mRFP)–
AtVam3 in pollen grains, and found that the fluorescence
seems not to be localized to particular organelle membranes
such as the vacuolar membrane (Fig. 4I, J). Intriguingly, the
fluorescence of GFP–SYP124 and GFP–SYP125 localized to
the tip region of pollen tubes during pollen tube elongation,
not to the cytoplasm of pollen grains (Fig. 4B, D). The localizations of GFP–SYP124 and GFP–SYP125 in growing pollen
tubes are a little bit different. Namely, GFP–SYP125 localized
to the apical region of the pollen tube, while GFP–SYP124
localized to the subapical region of the tip. In contrast, the
fluorescence of GFP–SYP131 and GFP–SYP132 localized
uniformly to the PMs not only of pollen tubes but also of
pollen grains (Fig. 4F, H).
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Expression profiles of SYP1 genes in embryonic cells
We examined the expression patterns of GFP–SYP1s during
embryonic development. Only SYP111 and SYP132 were
expressed in embryos at any stage of development. GFP–
SYP111 was highly expressed in leaf primordia cells (Fig. 5A),
probably due to the high dividing frequency of the cells in this
region. Strong GFP fluorescence was observed in the forming
cell plates, while relatively weak fluorescence was observed
in the mature PM (Fig. 5C). Meanwhile, GFP–SYP132 showed
constitutive expression throughout the development of
Fig. 4 Localizations of fluorescent-tagged SYP1s in pollen grains and
elongating pollen tubes. Mature pollen grains were scattered onto
coverslips from anthers of the transgenic plants at flowering stage
14. These pollen grains were isolated from the transgenic plants
expressing GFP–SYP124 (A), GFP–SYP125 (C), GFP–SYP131 (E),
GFP–SYP132 (G) and mRFP–AtVam3 (I and J). The localization of
each GFP-tagged SYP1 during pollen tube elongation is shown for
GFP–SYP124 (B), GFP–SYP125, (D) GFP–SYP131 (F) and GFP–SYP132
(H). Asterisks indicate the pollen grains that are lacking fluorescence,
because the transgenic plants expressing GFP–SYP131 and mRFP–
AtVam3 were hemizygous. Bars = 20 µm.
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Fig. 5 SYP111 and SYP132 were expressed in developing embryos.
Immature embryos were isolated from their seed coats, and GFP
fluorescence of embryos was observed with confocal microscopy. GFP
fluorescence of confocal sections (A, C, E) and corresponding DIC
(B, D, F) images represent mid-torpedo embryos expressing GFP–
SYP111 (A–D) and late heart embryos expressing GFP–SYP132 (E–F).
Bars = 40 µm (A) and 20 µm (C, E).
embryos, and was localized uniformly on the PM of all
embryonic cells (Fig. 5E). No other SYP1s were expressed
during the development of embryos (data not shown).
Discussion
Our gene expression analysis of PM Qa-SNAREs reveals that
these SNAREs have different expression profiles during different developmental stages. Analysis using microarray data
restricts the resolution of expression pattern analysis to the
organ level; the FTFLP procedure, in contrast, allows us to
analyze tissue specificities at a resolution of the level of one
cell and to observe the polarized distributions of these
SNAREs on the PM simultaneously.
In our analysis, no expression of GFP–SYP112 was
observed in any tissues examined, probably because SYP112
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has a low expression level under normal conditions (Supplementary Fig. S1). Although SYP112 is the closest paralog of
SYP111 and can completely complement the knolle phenotype when this transgene is expressed under the control of a
SYP111 promoter (Muller et al. 2003), it is likely to be latent
under normal growth conditions.
SYP121 and SYP122 are the closest paralogs among the
PM Qa-SNAREs, and both belong to the PEN1/ROR2 subgroup (Sanderfoot 2007). SYP121/PEN1 was first reported as
Nt-SYR1, a syntaxin involved in ABA-related ion channel
response in tobacco (Leyman et al. 1999), and was later
proposed to mediate pre-invasive immunity against the
non-host barley powdery mildew B. graminis in Arabidopsis
(Collins et al. 2003). SYP122 has also been reported to be
responsive to pathogen infection. syp121 syp122 double
mutants exhibit a dwarfed and necrotic growth phenotype
(Assaad et al. 2004). Recently, these two Qa-SNAREs were
reported to function as negative regulators of defense signaling pathways, including the salicylic acid, jasmonic acid and
ethylene signaling pathways (Zhang et al. 2007). In addition,
each has a different relationship with the tomato Rab11
homolog (Rehman et al. 2008). These data suggest that the
two syntaxins have overlapping functions not only in pathogen immunity but also in the general secretion pathway in
plants. The present study supports this hypothesis by showing that, while the localization and expression profiles of
SYP121 and SYP122 were almost identical in mature tissues,
their expression patterns in developing root tissues were
slightly different: SYP121 was expressed in the epidermal
cells and lateral root cap cells in the root tip, while SYP122
was expressed in the endodermal cells in the differentiation
zone; these observations were almost consistent with
available microarray data (see Supplementary Fig. S1). This
difference in expression may suggest that these two
Qa-SNARE molecules have different functions in early root
development at distinct cell layers.
SYP123 was specifically expressed in the root hair cells
and showed focal accumulation to the tip region of the
growing root hairs. Root hairs and pollen tubes typically
exhibit a polarized cell expansion known as tip growth.
Polarized vesicle trafficking to the tip region of growing root
hairs or pollen tubes is essential for this process (Xu and
Scheres 2005, Šamaj et al. 2006). Although small GTPases of
the Rab, Arf and Rho/Rac families (as well as their regulatory
proteins) have been shown to participate in tip growth
(Campanoni and Blatt 2007, Cheung and Wu 2008), so far no
SNARE molecules have been reported to be involved in this
growth. This is the first report that SYP123 may be involved
in the tip growth of root hairs.
Another significant observation we made was that SYP124
and SYP125 are expressed in pollen grains only. We found
that these SNAREs did not show clear localization on the
PM; rather they were dispersed thoughout the cytoplasm.
Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197 © The Author 2008.
Spatiotemporal expression control of PM SNAREs
This localization pattern might indicate that these SNAREs
exist on transport vesicles, rather than on any particular
organelle. Yet Qa-SNAREs are generally described as being
preferentially localized on the target membrane compartments, not on transport vesicles. This discrepancy must be
resolved in further examinations.
In summary, SYP132 is the only SYP1 family molecule that
is expressed constitutively in any of the tissues we examined.
In pollen, however, its GFP signal was relatively low compared with that of SYP131, a pollen-specific SYP13 subgroup
component; this observation is supported by the published
microarray data (Supplementary Fig. S1). These findings
may indicate the function of the SYP13 subgroup, namely its
general role in constitutive membrane trafficking to the PM,
specifically in the transport of housekeeping molecules, in all
plant cell types, with SYP132 predominating in vegetative
tissues. Other PM-resident Qa-SNAREs, on the other hand,
may have diverse functions depending on the type of membrane trafficking required in each cell type, with partial
redundancy. In particular, we found in this study that SYP123
is specifically expressed in root hair cells and accumulates at
the tip region of root hairs (Fig. 6). Our present study
strongly implies that multiple membrane trafficking routes
to the PM exist in the same and/or different cell types in an
arrangement which confers complexity to the process of
plant development. Recently, it has been reported that the
R-SNARE VAMP721/722 associates with SYP121/PEN1 and
SNAP33 to form a SNARE complex during plant immune
responses (Kwon et al. 2008). In future studies, it will be
necessary to determine which SNARE complexes are involved
in each of the membrane trafficking steps to the PM.
Materials and Methods
Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia (Col-0) seeds were
surface sterilized and grown on Jiffy-7 peat pellets for
appropriate periods under continuous light at 23°C. For the
observation of roots, seeds were placed on MS–agar plates
(2.1 g l–1 Murashige–Skoog salt, 1% sucrose, 1.5 mg l–1 thiamine,
2.5 mg l–1 nicotinic acid, 0.25 mg l–1 pyridoxine and 1.5% agar,
pH 5.8). After vernalization for 4 d, seeds were germinated
under the conditions described above, on vertically oriented
plates.
Plasmid construction and plant transformation
The translational fusions between GFP and the SYP1s were
generated using the FTFLP method described by Tian et al.
(2004) with some modifications. Briefly, for each SYP1, about
2,300 bp of an upstream region sequence with 5′-CACC, an
sGFP(S65T) sequence with a GGSG linker, and an SYP1 open
reading frame (ORF) followed by 1,000 bp of a downstream
Pollen-grain
SYP124
SYP132
SYP125
SYP131
Flowering stem
(epidermis)
SYP121
SYP123
Rosette leaf
(pavement cells)
SYP121
SYP122
SYP123
Ubiquitous
expression
(endodermis)
mature root
SYP123
(root hair)
root hair
elongation
SYP111
(phragmoplast)
root tip
(epidermis)
SYP122
SYP121
Fig. 6 Schematic illustration of the expression profiles of Arabidopsis
PM-Qa SYP1s. SYP132 was constitutively expressed in all tissues
examined in this study.
sequence were each amplified separately by PCR. Then the
three DNA fragments were conjugated in order, using the
triple-template PCR (TT-PCR) method to form a single DNA
fragment which carries the SYP1 ORF with the GFP sequence
at the 5′ end in tandem. In the case of mRFP–AtVam3, about
3,200 bp of an upstream region sequence followed by an
mRFP sequence was used for its construction. Whole constructs were designed to express the SNARE molecule fused
with fluorescent protein at the N-terminus. The fragment
was subcloned into pENTR/D-TOPO (Invitrogen, Carlsbad,
CA, USA), sequence verified, and then transferred into a
binary vector pGWB1 (Nakagawa et al. 2007) according
to the Gateway method. The sequences of primers used to
generate constructs are listed in Supplementary Table S1.
These constructs were introduced into Agrobacterium
tumefaciens strain C58 Rifr/pGV2260; subsequently, Arabidopsis wild-type plants (Col-0) were transformed by the
floral dipping method (Clough and Bent 1998). Screening of
transgenic plants was performed on MS plates containing
50 µg ml–1 hygromycin. T2 lines which showed a segregation
Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197 © The Author 2008.
287
K. Enami et al.
ratio of 3 : 1 for antibiotic resistance were used for further
experiments.
Confocal microscopy
GFP fluorescence signals and differential interference contrast (DIC) images were obtained using the Nikon ECLIPSE
E600 laser scanning microscope equipped with the C1siready confocal system (Nikon, Tokyo, Japan) and an argon
laser. The endocytosis marker FM 4–64 (Molecular Probes,
Eugene, OR, USA) was treated for 1 h before observation at a
final concentration of 30 µM, and 1 g l–1 of propidium iodide
solution was used to stain the cell wall. Pollen germination was
performed as described previously (Bovid and McCormick
2007). The collected images were processed using Nikon
EZ-C1 software.
3D analysis of GFP–SYP1 distribution
To examine the distribution of GFP–SYP1 in 3D, a 3D surface
model of a root hair was reconstructed from a series of confocal images and the intensities of GFP signals were mapped
on the reconstructed model. As a preprocessing step to
reduce the noise, a Gaussian filter was applied using ImageJ
1.39J (http://rsb.info.nih.gov/ij/). The 3D reconstruction and
intensity mapping was performed according to the technique employed by Kutsuna and Hasezawa (2005) using
ReantLight software (http://hasezawa.ib.k.u-tokyo.ac.jp/zp/
Kbi/ReantLight). Once the 3D models were reconstructed,
3D images were rendered interactively using ParaView 3.2.1
(http://www.paraview.org).
Analysis of microarray data
Microarray expression analysis data for nine PM Qa-SNAREs
were obtained using the Genevestigator V3 microarray database and analysis toolbox (Zimmermann et al. 2004, Hruz et al.
2008). The Genevestigator program is available on the web
site at (https://www.genevestigator.ethz.ch/gv/index.jsp).
Supplementary data
Supplementary data are available at PCP online.
Funding
Monbukagakusho (the Japanese Ministry of Education,
Culture, Sport, Science and Technology) grant-in-aid for
Basic Science Research (C) and a grant-in-aid for Scientific
Research on Priority Areas (to M.H.S.); the Yamada Science
Foundation (to M.H.S.); Japan Society for the Promotion of
Science (JSPS) Research Fellowships for Young Scientists
grant-in-aid (to T.U.); Nara Institute of Science and Technology
(supported by the Ministry of Education, Culture, Sports,
Science and Technology of Japan) grant-in-aid for Scientific
Research for Plant Graduate Students (to K.E.).
288
Acknowledgments
We thank Dr. G. Jürgens for critical reading of our manuscript. We also thank Dr. Y. Niwa for generously providing
the sGFP vector.
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(Received September 28, 2008; Accepted December 15, 2008)
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