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Selective Regulation of Maize Plasma Membrane Aquaporin
Trafficking and Activity by the SNARE SYP121
W
Arnaud Besserer,a Emeline Burnotte,a Gerd Patrick Bienert,a Adrien S. Chevalier,a Abdelmounaim Errachid,a
Christopher Grefen,b Michael R. Blatt,b and François Chaumonta,1
a Institut
des Sciences de la Vie, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium
of Plant Physiology and Biophysics, Institute of Molecular, Cell, and Systems Biology, University of Glasgow,
Glasgow G12 8QQ, United Kingdom
b Laboratory
Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We
previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic
reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated
by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma
membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K+ channels. We demonstrate that the
expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to
a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5
water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo
(bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches
allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of
SYP121 in regulating K+ channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the
regulation of the cell osmotic homeostasis.
INTRODUCTION
Water uptake and maintenance of the cell osmotic homeostasis
are major constraints encountered by living organisms. One of
the protein families involved in the regulation of these processes
is the aquaporin family. Aquaporins are small (28 to 34 kD)
membrane proteins that form tetrameric channels facilitating the
movement of water and/or small neutral solutes through cellular
membranes (Gomes et al., 2009). Compared with mammals,
plants express a high number of aquaporin isoforms, underlining
their fundamental roles in growth, development, and abiotic
stress tolerance (Maurel et al., 2008; Gomes et al., 2009; Heinen
et al., 2009). Among the plant aquaporins, the plasma membrane intrinsic proteins (PIPs) are subdivided into two sequencerelated groups, PIP1s and PIP2s, the members of which exhibit
different water channel activities when expressed in Xenopus
laevis oocytes (Chaumont et al., 2000; Temmei et al., 2005;
Vandeleur et al., 2009; Bellati et al., 2010; Otto et al., 2010).
When expressed alone, maize (Zea mays) PIP1s are inactive,
whereas Zm-PIP2s cause a marked increase in the osmotic
water permeability coefficient, Pf. However, when coexpressed,
Zm-PIP1s and Zm-PIP2s physically interact to increase the Pf
compared with oocytes expressing Zm-PIP2s alone (Fetter
et al., 2004).
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: François Chaumont
([email protected]).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.112.101758
As integral membrane proteins, PIPs are synthesized in the
endoplasmic reticulum (ER) and then transported along the secretory pathway to reach the plasma membrane. We previously
showed that PIP trafficking is regulated by a heterooligomerization process (Fetter et al., 2004; Zelazny et al., 2007). When
transiently expressed alone in maize cells, Zm-PIP1s and ZmPIP2s differ in their subcellular localization. Zm-PIP1s are retained in the ER, whereas Zm-PIP2s are targeted to the plasma
membrane (Zelazny et al., 2007). However, upon coexpression,
Zm-PIP1s are relocalized from the ER to the plasma membrane
as a result of their physical interaction with Zm-PIP2s, demonstrated by Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy experiments. A diacidic
motif, DIE (Asp-Ile-Glu) at position 4 to 6 in the N terminus of
Zm-PIP2;5 is essential for ER export (Zelazny et al., 2009). This
motif is conserved and functional in Zm-PIP2;4 but is absent in
Zm-PIP2;1. However, the replacement of the N terminus of ZmPIP1;2 by the N terminus of Zm-PIP2;5 containing the diacidic
motif is not sufficient to cause the export of Zm-PIP1;2 from the
ER. A functional diacidic motif has also been identified in the N
terminus of Arabidopsis thaliana PIP2;1 (Sorieul et al., 2011). In
addition, the traffic and plasma membrane abundance of the AtPIP2;1 is regulated by different posttranslational modifications,
including phosphorylation and ubiquitylation (Prak et al., 2008;
Lee et al., 2009). Once located in the plasma membrane, AtPIP2;1 can move into or out of membrane microdomains and
undergoes constitutive cycling, involving clathrin-dependent and
raft-associated endocytosis (Paciorek et al., 2005; Dhonukshe
et al., 2007; Li et al., 2011). The presence of PIPs in endosomes
reflects a dynamic regulation of their density at the plasma
membrane and their sorting between storage or degradation
The Plant Cell Preview, www.aspb.org ã 2012 American Society of Plant Biologists. All rights reserved.
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paths (Wudick et al., 2009; Luu et al., 2012). In this process, the
trans-Golgi network (TGN) could act as a late sorting platform in
the secretory pathway (Robinson et al., 2008; Richter et al.,
2009), but the molecular actors regulating the post-Golgi trafficking of PIPs and their insertion in the plasma membrane remain elusive.
In mammals, the proper trafficking and anchoring of aquaporin2 (AQP2) in the apical membrane of collecting duct epithelial cells are specifically regulated by SNARE (for soluble
N-ethylmaleimide-sensitive factor protein attachment protein
receptor) complexes (Mistry et al., 2009; Wang et al., 2010).
SNAREs are membrane proteins that facilitate vesicular
trafficking in eukaryotes. They are subdivided into Q- and RSNAREs, according to their conserved residues within the
SNARE motif (Fasshauer et al., 1998). SNAREs are present in the
membranes of all subcellular compartments and play a major
role in vesicular fusion and membrane biosynthesis. Genome
analysis showed that the SNARE family is larger in plants than in
other eukaryotes (Sanderfoot, 2007). Indeed, more than 60
SNARE isoforms have been identified in the Arabidopsis genome, and orthologs are found in rice (Oryza sativa) for most of
them, supporting the idea that they are conserved between
monocots and dicots (Sutter et al., 2006a). Syntaxins belong to
the Q-SNARE subfamily and, in Arabidopsis, seven of them localize to the plasma membrane and are involved in traffic at this
membrane (Grefen et al., 2011). Among these plasma membrane resident syntaxins, SYP121 has been the most intensively
studied and is known to mediate vesicle traffic between the
Golgi complex and plasma membrane (Geelen et al., 2002).
Disrupting its function by overexpressing a dominant-negative
cytosolic (so-called Sp2) fragment specifically affected the
trafficking to the plasma membrane (Tyrrell et al., 2007). In tobacco (Nicotiana tabacum), SYP121-Sp2 suppressed the mobility and delivery of the potassium channel KAT1 to the plasma
membrane, but not of the H+-ATPase (Sutter et al., 2006b). Interestingly, SYP121 was recently shown to regulate the gating of
the AKT1/KC1 K+ channel complex through a direct interaction
involving an FxRF motif located within the first 12 residues of the
protein (Honsbein et al., 2009; Grefen et al., 2010). Altogether,
these data raise the hypothesis that SYP121 acts as a molecular
governor, coordinating the gating of ion channels in parallel with
membrane traffic and, therefore, ion uptake with increased
membrane surface area through vesicle fusion during cell expansion (Grefen and Blatt, 2008; Honsbein et al., 2011).
As water movement is a key element of the mechanism that
regulates cell osmotic homeostasis, we wondered if SYP121
could also regulate the traffic and activity of PIP aquaporins. In
barley (Hordeum vulgare), PIP2 colocalized in ROR2/SYP121
vesicle-like compartments, supporting this hypothesis (Kwaaitaal
et al., 2010). We report here that the SYP121-Sp2 from Arabidopsis and its putative ortholog from maize affect the delivery of
Zm-PIP2;5 to the plasma membrane in maize mesophyll protoplasts and epidermal cells. Furthermore, the traffic inhibition is
correlated with a decrease in the protoplasts Pf. As Zm-PIP2;5
water channel activity is also reduced in the presence of the
ZmSYP121-Sp2 fragment in Xenopus oocytes, we tested the
possibility that both proteins directly interact. Physical interaction
between both proteins was demonstrated in vitro and in vivo by
different technical approaches. These data indicate that SNARE
might play an important role in the regulation and maintenance of
osmolarity in the cytosol through a coordinated regulation of
aquaporins and K+ channels.
RESULTS
The AtSYP121-Sp2 Fragment Reduces Plasma Membrane
Delivery of Zm-PIP2;5
When expressed in maize mesophyll protoplasts, monomeric
yellow fluorescent protein (mYFP):ZmPIP2;5 accumulates in the
plasma membrane (Zelazny et al., 2007). To determine whether
this localization is dependent on the activity of the syntaxin
SYP121 or not, we coexpressed mYFP:ZmPIP2;5 and the Arabidopsis dominant-negative AtSYP121-Sp2 fragment fused to
the C terminus of monomeric cyan fluorescent protein (mCFP)
and analyzed the cellular localization and intensity of the fluorescent proteins using confocal laser scanning microscopy
(CLSM). Fusions of the fluorescent proteins to the N terminus of
At-SYP121 and Zm-PIP2;5 have been previously shown not to
affect the subcellular localization of both proteins in protoplasts
(Uemura et al., 2004; Zelazny et al., 2007). When expressed
alone, mYFP:ZmPIP2;5 mainly accumulated in the cell periphery
(Figure 1A). The fluorescent signal colocalized with the styryl dye
FM4-64 (Figures 1Aa to 1Ac) and mCFP:tagged Np-PMA2
H+-ATPase (Lefebvre et al., 2004) (Figures 1Ad to 1Af), demonstrating that ZmPIP2;5 was mainly present in the plasma
membrane. When coexpressed with mCFP:AtSYP121-Sp2,
mYFP:ZmPIP2;5 was still present in the plasma membrane, but
the fluorescent signal intensity was much weaker than in cells
expressing mYFP:PIP2;5 alone (Figures 1Ag to 1Ai). mYFP:
ZmPIP2;5 also labeled internal structures. To quantify the plasma
membrane fluorescence intensity, an image analysis procedure
was developed. Briefly, the average thickness of the plasma
membrane was determined using the colocalized mYFP:
ZmPIP2;5 and FM4-64 fluorescent signals, and the difference
between the overall and intracellular fluorescent signals was used
to calculate the plasma membrane fluorescence (for more details,
see Methods). The mYFP plasma membrane fluorescence intensity
was significantly lower (P < 0.05) in protoplasts coexpressing the
mYFP:ZmPIP2;5 and mCFP:AtSYP121-Sp2 fragment than in protoplasts expressing mYFP:ZmPIP2;5 alone (Figure 1B), indicating
that AtSYP121-Sp2 interferes with Zm-PIP2;5 delivery to the
plasma membrane.
Zm-SYP121 Is the Functional Ortholog of At-SYP121
Maize database analysis allowed the identification of a maize
SYP121 protein that shared 54% amino acid identity with AtSYP121. Both proteins cluster in the same phylogenetic group
as shown by the neighbor-joining tree (see Supplemental Figure
1 and Supplemental Data Set 1 online). To test if this sequence
similarity could reflect a conservation in the regulation of ZmPIP2;5 delivery to the plasma membrane, the SYP121 cDNA was
cloned from total RNA extracted from maize roots and fused
to mCFP and mYFP sequences in plant expression vectors. A
PIP Aquaporin Regulation by SNARE
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Figure 1. Both AtSYP121-Sp2 and ZmSYP121-Sp2 Fragments Selectively Reduce the Accumulation of Zm-PIP2;5 in the Plasma Membrane of Maize
Mesophyll Protoplasts.
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truncated version of the cDNA was also prepared to produce the
ZmSYP121-Sp2 fragment. These constructs were then transfected in maize mesophyll protoplasts. Figures 1C and 1D show
the subcellular localization of mYFP:ZmPIP2;5 and the plasma
membrane fluorescence signal intensity, respectively, in protoplasts transiently expressing mYFP:ZmPIP2;5 alone (Figures
1Ca and 1Cb) and coexpressing mYFP:ZmPIP2;5 and mCFP:
ZmSYP121 (Figures 1Ce and 1Cf) or mCFP:ZmSYP121-Sp2
(Figures 1Cg and 1Ch). The soluble mCFP was coexpressed
with mYFP:ZmPIP2;5 as a control (Figures 1Cc and 1Cd). When
expressed alone or coexpressed with mCFP or mCFP:
ZmSYP121, mYFP:ZmPIP2;5 accumulated in the cell plasma
membrane (Figures 1Cb, 1Cd, and 1Cf). Quantification of the
plasma membrane fluorescence intensities in the mYFP channel
showed that the mYFP:ZmPIP2;5 and mCFP:ZmSYP121 coexpressing protoplasts had a slightly weaker plasma membrane
fluorescence than protoplasts expressing mYFP:ZmPIP2;5 alone,
but the intensities were not significantly different (Figure 1D). By
contrast, upon coexpression with the mCFP:ZmSYP121-Sp2
fragment, the mYFP:ZmPIP2;5 plasma membrane signal intensity
was strongly decreased (Figures 1Ch and 2Af), indicating that
Zm-SYP121 is a functional ortholog of AtSYP121. In protoplasts
cotransfected with equal amounts of plasmids encoding mCFP:ZmSYP121-Sp2 or mYFP:ZmPIP2;5, mYFP:ZmPIP2;5 only poorly
accumulated in internal structures. However, when protoplasts
were transfected with a 3:1 ratio of mCFP:ZmSYP121-Sp2 and
mYFP:ZmPIP2;5 plasmids, a significant accumulation of mYFP:
ZmPIP2;5 in internal structures was observed (see Supplemental
Figure 2 online).
For comparison, we examined the fluorescence distribution of
mCFP:NpPMA2 expressed in maize protoplasts. It was previously shown that the delivery of Np-PMA2 to the plasma
membrane was not affected by AtSYP121-Sp2 in tobacco epidermal cells (Sutter et al., 2006a). When coexpressed with
mYFP:ZmSYP121-Sp2 in maize protoplasts, no modification in
mCFP:NpPMA2 plasma membrane localization and intensity
was detected (Figures 1E and 1F).
To analyze the dynamics of the effect of ZmSYP121-Sp2 on
newly synthesized mYFP:ZmPIP2;5 that reaches the plasma
membrane via anterograde transport, we compared the intensity
of mYFP signal according to time in mesophyll protoplasts
expressing mYFP:ZmPIP2;5 alone or coexpressing mYFP:
ZmPIP2;5 with mCFP:ZmSYP121-Sp2. One image acquisition
was performed every 30 min during 16 h after cell transfection
(see Supplemental Figure 3 online with associated Supplemental
Movie 1 and Supplemental Movie Legend 1 online). The quantification of the global (plasma membrane and intracellular) mYFP
fluorescent signals in protoplasts expressing mYFP:ZmPIP2;5
alone or in combination with mCFP:ZmSYP121-Sp2 is shown
in Supplemental Figure 4 online. The mYFP and mCFP signals
were detected from 5 to 8 h after transfection. During the first 8
h, the increase in mYFP signal intensity (integrated density)
was slightly lower but not significantly different in cells coexpressing mYFP:ZmPIP2;5 and mCFP:ZmSYP121-Sp2 compared with protoplasts expressing mYFP:ZmPIP2;5 alone,
indicating that the protein synthesis rate was not affected.
However, 12 h after transfection, while the mYFP signal was
still increasing in protoplasts expressing mYFP:ZmPIP2;5
alone, it plateaued in cells coexpressing mCFP:ZmSYP121Sp2 and mYFP:ZmPIP2;5. During this time range, the signal of
mCFP:ZmSYP121-Sp2 was still rising, demonstrating that
protein synthesis was not affected by mCFP:ZmSYP121-Sp2
expression.
To monitor the cycling of Zm-PIP2;5 from intracellular compartments to the plasma membrane or within the membrane
surface, we performed fluorescence recovery after photobleaching (FRAP) experiments in tobacco epidermal cells transiently expressing mYFP:ZmPIP2;5 alone or together with
mCFP:ZmSYP121-Sp2 (see Supplemental Figure 5 online). In
cells expressing mYFP:ZmPIP2;5 alone, a recovery of 20% of
Figure 1. (continued).
(A) Plasma membrane abundance of mYFP:ZmPIP2;5 in maize mesophyll protoplasts. (a) to (c) Protoplast expressing mYFP:ZmPIP2;5 (a). (b) Channel
showing the FM4-64 fluorescence and chlorophyll a autofluorescence. (c) Merged image. (d) to (f) Protoplast coexpressing mYFP:PIP2;5 (d) and
mCFP:PMA2 (e). (f) Merged image. (g) to (i) Protoplast coexpressing mYFP:PIP2;5 (g) and mCFP:AtSYP121-Sp2 (h). (i) Merged image. Bars = 5 µm.
(B) Quantification of mYFP:ZmPIP2;5 fluorescence intensity in the protoplast plasma membrane (PM). Acquisitions were performed with the same
settings for all samples. Each bar represents the mean of five independent experiments (10 protoplasts per experiment). Error bars show the 95%
confidence interval. Student’s t test, P < 0.05 was used to compare the data. Means that are significantly different (P < 0.05) are indicated by different
letters. a.u., arbitrary units.
(C) Plasma membrane abundance of Zm-PIP2;5 in maize mesophyll protoplasts. (a) and (b) Protoplast expressing mYFP:ZmPIP2;5 (b). (a) Chlorophyll
a autofluorescence. (c) and (d) Protoplast coexpressing the soluble mCFP (c) and mYFP:ZmPIP2;5 (d). (e) and (f) Protoplast coexpressing mCFP:
ZmSYP121 (e) and mYFP:ZmPIP2;5 (f). (g) and (h) Protoplast coexpressing mCFP:ZmSYP121-Sp2 (g) and mYFP:ZmPIP2;5 (h). All the pictures were
acquired with the same settings. In panel (a), the chloroplast autofluorescence is shown, whereas, in panels (c), (e), and (f), it is subtracted. Bars = 5µm.
(D) Quantification of mYFP:ZmPIP2;5 fluorescence intensity in the protoplast plasma membrane. Each bar represents the mean of three independent
experiments (10 protoplasts by experiments). Error bars show 95% confidence interval. Statistical differences were inferred by analysis of variance
(ANOVA) followed by Tukey’s honestly significant difference test. Means that are significantly different (P < 0.05) are indicated by different letters.
(E) Plasma membrane abundance of mCFP:PMA2 in maize mesophyll protoplasts. (a) to (c) Protoplast expressing mCFP:PMA2 (a). (b) YFP channel. (c)
Chlorophyll a channel. (d) to (f) Protoplast coexpressing mCFP:PMA2 (d) and mYFP:ZmSYP121-Sp2 (e). (f) Chlorophyll a channel. All the images were
acquired with the same settings. Bars = 5 µm.
(F) Quantification of mCFP:PMA2 fluorescence intensity in the protoplast plasma membrane. Each bar represents the mean of three independent
experiments (10 protoplasts per experiment). Error bars show the 95% confidence interval. Means are not significantly different (Student’s t test, P =
0.90).
PIP Aquaporin Regulation by SNARE
the initial plasma membrane fluorescence was detected 150 s
after photobleaching. These data are in agreement with previous
findings on the KAT1 K+ channel in Arabidopsis (Honsbein et al.,
2009) and on At-PIP2;1 (Sorieul et al., 2011; Luu et al., 2012). In
cells coexpressing mCFP:ZmSYP121-Sp2 and mYFP:ZmPIP2;5,
the recovery reached only 5% of the initial plasma membrane
signal.
The Inhibition of Zm-PIP2;5 Delivery to the Plasma
Membrane Is SYP121-Sp2 Specific
To test whether the delivery of Zm-PIP2;5 to the plasma membrane was specifically dependent on SYP121, mYFP:ZmPIP2;5
was coexpressed in maize protoplasts with the Sp2 truncated
forms of At-SYP71, At-SYP122, or At-SYP21 syntaxins, and its
abundance in the plasma membrane was measured (Figure 2).
SYP71 and SYP122 are syntaxins localized in the plasma
membrane (Leyman et al., 2000; Borner et al., 2005; Suwastika
et al., 2008), while SYP21 was found in the prevacuolar compartment membrane (da Silva Conceição et al., 1997; Sanderfoot
et al., 2001; Tse et al., 2004). None of these Sp2 fragments
affected the plasma membrane accumulation of mYFP:
ZmPIP2;5 (Figures 2A and 2B), although AtSYP21-Sp2 has been
shown to be active in suppressing traffic to the (pre)vacuolar
compartment (Tyrrell et al., 2007). The plasma membrane fluorescent signal intensity detected in cells coexpressing mCFPtagged Sp2 fragments of At-SYP71, At-SYP122, or At-SYP21
with mYFP:ZmPIP2;5 was similar to the intensity found in protoplasts expressing mYFP:ZmPIP2;5 alone or in combination
with mCFP:ZmSYP121. As previously mentioned, protoplasts
coexpressing mYFP:ZmPIP2;5 and mCFP:ZmSYP121-Sp2
showed a strong reduction of the plasma membrane signal.
These data demonstrate that the regulation of Zm-PIP2;5 trafficking to the plasma membrane is specifically dependent on
Zm-SYP121.
Coexpression of Zm-PIP2;5 and the ZmSYP121-Sp2
Fragment Leads to a Decrease in Maize Protoplast Pf
and Reproduces the Cell Water Permeability Phenotype
of the syp121-1 Arabidopsis Mutant
To determine whether the reduction in Zm-PIP2;5 delivery to the
plasma membrane was correlated with a decrease in the membrane water permeability coefficient, we analyzed the swelling of
protoplasts expressing mYFP:ZmPIP2;5 or mCFP:ZmSYP121
singly or coexpressing mYFP:ZmPIP2;5 with mCFP:SYP121 or
mCFP:ZmSYP121-Sp2. Mock protoplasts were used as a negative control. For each protoplast population, the Pf values were
obtained as described (Moshelion et al., 2004; Volkov et al.,
2007), plotted on a frequency diagram (Figure 3A), and the
medians were calculated (Figure 3B). Expression of mYFP:
ZmPIP2;5 alone induced an important increase in the Pf values
compared with the mock protoplasts. Its coexpression with
mCFP:ZmSYP121 did not alter this high Pf. For both protoplast
populations, the Pf distributions were similar, with the presence
of cells having a Pf higher than 10 µm s21. By contrast, protoplasts coexpressing mYFP:ZmPIP2;5 and mCFP:ZmSYP121Sp2 exhibited similar Pf values as mock or mCFP:ZmSYP121
expressing protoplasts.
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A detailed analysis of the swelling curves obtained with protoplasts expressing mYFP:ZmPIP2;5 or mCFP:ZmSYP121-Sp2
and mYFP:ZmPIP2;5 was performed as previously reported
(Moshelion et al., 2004; Volkov et al., 2007; Moshelion et al.,
2009). While no difference in the lag phase preceding the
swelling event was detected, the Pf values determined 15, 30,
and 45 s after the solution exchange increased during the
swelling event but were always significantly lower for protoplasts coexpressing mYFP:ZmPIP2;5 and mCFP:ZmSYP121Sp2 compared with their respective controls (see Supplemental
Figure 6 online). In addition, the Pf differences between these
two cell populations increased with the swelling time, probably
reflecting differences in the amount of active aquaporins reaching
the plasma membrane (Moshelion et al., 2004).
To further characterize the role of SYP121 in regulating the
membrane water permeability, leaf protoplasts isolated from the
wild type (Columbia-0) and syp121-1 Arabidopsis mutated line
(Collins et al., 2003) were isolated and subjected to an osmotic
challenge, and the Pf was determined. Interestingly, syp121-1
protoplasts had a lower Pf than wild-type ones (Figures 3C and
3D), phenocopying the permeability of maize protoplasts expressing the SYP121-Sp2 fragment. These data demonstrate
a clear link between SYP121 function in Arabidopsis and the
regulation of the plasma membrane water permeability controlled by aquaporins.
Finally, we note that no significant increase in Pf was measured
in maize protoplasts coexpressing mYFP:ZmPIP2;5 and mCFP:
ZmSYP121-Sp2 compared with mock cells (Figure 3B), even if
some mYFP fluorescence was detected in the plasma membrane
(Figures 1Ch and 2Af). These observations led us to question
whether ZmSYP121-Sp2 might regulate Zm-PIP2;5 activity independently of any effect mediated through trafficking.
The Water Channel Activity of Zm-PIP2;5 Is Regulated
by ZmSYP121-Sp2 in Xenopus Oocytes
We tested the impact of Zm-SYP121 and ZmSYP121-Sp2 on
the water channel activity of Zm-PIP2;5 expressed in Xenopus
oocytes. Venus:ZmPIP2;5 was expressed alone or together
with Zm-SYP121 or the ZmSYP121-Sp2 fragment fused to a 6
His tag at the N terminus. The Pf values calculated from oocyte
swelling experiments were plotted in Figure 4A. In contrast
with 6His:ZmSYP121, 6His:ZmSYP121-Sp2 induced a significant decrease in the Pf of Zm-PIP2;5 expressing oocytes, but
the Pf was significantly higher than the Pf of mock oocytes and
oocytes expressing 6His:ZmSYP121 or 6His:ZmSYP121-Sp2
alone. The Pf decrease might be due to impairment of the
delivery of Venus:ZmPIP2;5 into the oocyte plasma membrane.
To determine the amount of protein in the cell membrane, the
Venus fluorescence was detected in fixed oocytes using CLSM
and quantified (Figures 4B and 4C). When coexpressed
with either 6His:ZmSYP121 or 6His:ZmSYP121-Sp2, Venus:
ZmPIP2;5 was still localized in the plasma membrane (Figures
4Ba, 4Bb, and 4Bc) and the plasma membrane Venus fluorescence intensity was similar to the intensity measured in the
plasma membrane of oocytes expressing Venus:ZmPIP2;5
alone (Figure 4C). Immunodetection analysis of the microsomal
fraction showed that the Zm-PIP2;5 amount was similar in
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oocytes expressing Zm-PIP2;5 alone or together with ZmSYP121 or Zm-SYP121-Sp2 (Figures 4D and 4E). As the Pf
values measured in these experiments were around 0.4 to 0.6
µm s21, far below the maximum values reported when higher
amounts of Zm-PIP cRNAs were injected, we can exclude the
possibility that the protein expression was saturated (Chaumont
et al., 2000; Fetter et al., 2004). Altogether, these data are
consistent with a direct effect of Sp2 on Zm-PIP2;5 activity that
is independent of any action on Zm-PIP2;5 trafficking to the
plasma membrane.
Zm-PIP2;5 and Zm-SYP121 Physically Interact in Xenopus
Oocytes and in Plant Cells
The regulation of the water channel activity of Zm-PIP2;5 by the
ZmSYP121-Sp2 fragment might be due to a direct physical interaction between both proteins. To evaluate this possibility, we
extracted the microsomes of oocytes expressing 6His-tagged
Zm-PIP2;5 alone or together with Venus:ZmSYP121 and purified
6His:ZmPIP2;5 on a nickel–nitrilotriacetic acid affinity column.
The presence of 6 His:ZmPIP2;5 and Venus:ZmSYP121 in the
elution fraction was checked by immunodetection using antibodies raised against ZmPIP2;5 and green fluorescent protein
(GFP), respectively (Chaumont et al., 2000; Hachez et al., 2006)
(Figures 5A and 5B). A signal corresponding to the Zm-PIP2;5 dimer
was detected in the eluted fractions coming from oocytes expressing 6His:ZmPIP2;5 alone or together with Venus:ZmSYP121
(Figure 5A, lanes a and b). No signal was observed in eluted fractions from mock oocytes or oocytes expressing Venus:ZmSYP121
alone (Figure 5A, lanes c and d). The 6His:ZmPIP2;5 dimeric form
was due to the presence of a disulfide bond linking two Zm-PIP2;5
monomers (Bienert et al., 2012) and was detected as no reducing
agent was added in the solubilization buffer. Interestingly, a YFP
signal corresponding to Venus:ZmSYP121 was observed in the
fraction coming from oocytes coexpressing 6His:ZmPIP2;5 and
Venus:ZmSYP121 (Figure 5B, lane b), demonstrating that both
proteins were copurified.
Figure 2. Inhibition of Zm-PIP2;5 Delivery to the Plasma Membrane Is
Specific for ZmSYP121-Sp2.
(A) Plasma membrane abundance of mYFP:ZmPIP2;5 in maize mesophyll protoplasts. (a) and (b) Protoplast expressing mYFP:ZmPIP2;5 (b).
(a) Chlorophyll a autofluorescence. (c) and (d) Protoplast coexpressing
mCFP:ZmSYP121 (c) and mYFP:ZmPIP2;5 (d). (e) and (f) Protoplast
coexpressing mCFP:ZmSYP121-Sp2 (e) and mYFP:ZmPIP2;5 (f). (g)
and (h) Protoplast coexpressing mCFP:AtSYP71-Sp2 (g) and mYFP:
ZmPIP2;5 (h). (i) and (j) Protoplast coexpressing mCFP:AtSYP21-Sp2 (i)
and mYFP:ZmPIP2;5 (j). (k) and (l) Protoplast coexpressing mCFP:AtSYP122-Sp2 (k) and mYFP:ZmPIP2;5 (l). All images were acquired with
the same settings. In panel (a), autofluorescence of the chlorophyll a is
shown, whereas in panels (c) to (k), chlorophyll a autofluorescence is
subtracted. Bars = 5 µm.
(B) Quantification of mYFP:ZmPIP2;5 fluorescence signal intensity in the
protoplast plasma membrane (PM). Each bar represents the mean of
three independent experiments (10 protoplasts per experiment). Error
bars show the 95% confidence interval. Statistical differences were inferred by ANOVA, followed by the Tukey’s honestly significant difference
test. Means that are significantly different (P < 0.05) are indicated by
different letters. a.u., arbitrary units.
PIP Aquaporin Regulation by SNARE
7 of 19
Figure 3. Maize Mesophyll Protoplasts Coexpressing mCFP:ZmSYP121-Sp2 and mYFP:ZmPIP2;5 Phenocopies the Lower Plasma Membrane Water
Permeability (Pf) of Arabidopsis syp121-1 Mesophyll Protoplasts.
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The Plant Cell
The interaction between Zm-PIP2;5 and Zm-SYP121 was
also checked in plant cells using bimolecular fluorescence
complementation (BiFC) experiments in maize epidermal cells
transfected by biolistic bombardment. We used split Venus
(ADE48838.1) fusion constructs (Nour-Eldin et al., 2006; Shyu
et al., 2006). First, the plasma membrane localization of transiently expressed mYFP:ZmPIP2;5 was confirmed by colocalization with the styryl dye FM4-64 (Figures 5Ca to 5Cc). When
mCFP:ZmSYP121 and mYFP:ZmPIP2;5 were coexpressed,
both proteins colocalized at the cell periphery (Figures 5Cd to
5Cg), as previously observed in maize protoplasts (Figures 1Ce
to 1Cf). As we previously showed that Zm-PIP2;5 and ZmPIP1;2 interact in maize cells (Zelazny et al., 2007), we used
them as a positive control in the BiFC experiments. When VenusN:ZmPIP2;5 and VenusC:ZmPIP1;2 were coexpressed,
a fluorescent signal was detected mostly in the cell periphery
(Figure 5Ch), validating the method. When VenusN:ZmPIP2;5
and VenusC:ZmSYP121 were coexpressed, a Venus signal was
observed in the cell plasma membrane (Figure 5Ci), demonstrating an interaction between the two proteins. Fluorescent
signals were also detected in the plasma membrane and/or internal membranes when these proteins were coexpressed in
maize mesophyll protoplasts (Figures 5Cl to 5Co). The spectral
specificity of the signal was validated by determination of its
emission spectrum (Figure 5D). Cells expressing VenusN:PMA2
and VenusC:ZmSYP121 were used as negative controls for interaction (Figures 5Cj and 5Ck or 5Cp and 5Cr). Similar BiFC
results were obtained when the proteins were expressed in tobacco epidermal cells (see Supplemental Figure 7 online).
To further validate the direct interaction between Zm-PIP2;5
and Zm-SYP121, FRET experiments were performed on maize
mesophyll protoplasts and tobacco epidermal cells coexpressing mCFP:ZmPIP1;2 and mYFP:ZmPIP2;5 or mCFP:ZmSYP121
and mYFP:ZmPIP2;5. The FRET efficiency was determined by
measuring the fluorescence intensity of the donor fluorochrome
before and after acceptor photobleaching (Bhat et al., 2005). A
representative experiment is given in Supplemental Figure 8
online for each cell type, and quantitative results are reported in
Table 1. Coexpression of mCFP:ZmPIP1;2 and mYFP:ZmPIP2;5
or mCFP:ZmSYP121 and mYFP:ZmPIP2;5 in maize protoplasts
gave a FRET efficiency of ;26 and 15%, respectively, values
significantly higher than those of the negative controls constituted by the coexpression of mCFP:ZmPIP2;5 and mYFP:ROP6
(Zelazny et al., 2007). These values were validated by assessing
FRET efficiency by acceptor-sensitized emission on the same
cells. Data obtained by the two methods showed 3% divergence,
which is comparable to the stochastic variations observed in the
acceptor photobleaching experiments (see Supplemental Figure 9
online).
Additionally, we tested the potential for SYP121 interaction
with Zm-PIP2;5 using a mating-based, split-ubiquitin assay,
much as previously described in the analysis of the Arabidopsis SYP121 interaction with the K+ channels KC1 and KAT1
(Honsbein et al., 2009, 2011; Grefen et al., 2010). In this case,
we used a ZmPIP2;5-Cub-PLV fusion protein as bait and tested
its ability to rescue growth of diploid yeast carrying the prey
fusions of Zm-SYP121 and At-SYP121 with Nub, the N-terminal
half of ubiquitin. For purposes of comparison, we tested for
interactions with a homolog of Zm-PIP2;5, the Arabidopsis
aquaporin At-PIP2;2, and we performed each assay with both
the maize SNARE and the Arabidopsis SNARE SYP121. Figures
6A and 6B summarize the results of these experiments, with the
column on the left in each frame showing the control for growth
on mating at 24 h and the subsequent three columns showing
growth on increasing concentrations of Met that repress the
expression of the bait. Figure 6C shows the immunoblot analysis
for the two experiments, confirming the expression of all four
proteins and the presence of bands at the correct molecular
weights. From these results, it is clear that Zm-PIP2;5 interacts
with the maize SYP121, and even more strongly with AtSYP121, in high concentrations of Met. The Arabidopsis aquaporin also interacted with At-SYP121 but showed only a weak
association with its maize homolog. We note that the negative
control (NubG) also showed a low level of growth rescue with
the ZmPIP2;5-Cub-PLV fusion, which may suggest some leakage, possibly a low level of proteolytic turnover in this case
sufficient to promote growth under the nonstringent, low Met
conditions. Nonetheless, a comparison shows a clear interaction of Zm-PIP2;5 with Zm-SYP121, consistent with our
copurification data. Taken together, these data demonstrate
a direct interaction between Zm-SYP121 and Zm-PIP2;5, consistent with a regulation of the water channel activity of ZmPIP2;5 by SYP121.
DISCUSSION
Aquaporins constitute an important selective pathway for water
and small neutral solutes to move across cellular membranes.
However, to exert their function, aquaporins have to reach their
target membrane and be active. PIP aquaporins are synthesized at
the ER and traffic through the secretory pathway before reaching
the plasma membrane (Paciorek et al., 2005; Dhonukshe et al.,
Figure 3. (continued).
(A) Relative distribution of Pf values of maize mesophyll protoplasts untransfected or (co)expressing mYFP:ZmPIP2;5, mYFP:ZmPIP2;5 and mCFP:
ZmSYP121, mYFP:ZmPIP2;5 and mCFP:SYP121-Sp2, or mCFP:ZmSYP121.
(B) Median Pf values for each protoplast subpopulation shown in (A).
(C) Relative distribution of Pf values of mesophyll protoplasts from wild-type (WT) and syp121 Arabidopsis plants.
(D) Median Pf values for both Arabidopsis protoplast subpopulations.
Each bar represents the mean of 20 measurements. Error bars show 95% confidence interval. Statistical differences were inferred by the Kruskall-Wallis
test followed by a Dunns test or Mann-Whitney U test. Medians that are significantly different (P < 0.05) are shown with different letters.
PIP Aquaporin Regulation by SNARE
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2007; Jaillais et al., 2007; Zelazny et al., 2007; Sorieul et al.,
2011). The fusion of vesicles coming from the endomembrane
compartments to the plasma membrane is generally dependent
on the activities of SNARE complexes in all eukaryotic cells
(Pratelli et al., 2004). Among the SNARE proteins, the plasma
membrane syntaxin SYP121 was demonstrated to regulate the
delivery but also the gating of plant K+ channels through direct
physical interaction, indicating that this protein may play an
important role in the control of ion transport and cellular volume
(Sutter et al., 2006b; Honsbein et al., 2009).
As aquaporin-facilitated water movement through the plasma
membrane is an inherent process controlling the cell water homeostasis, we questioned whether SNAREs also regulate the
traffic and activity of these channels. Our results now address
this question using Zm-PIP2;5 as a model aquaporin and the
plasma membrane–resident syntaxin SYP121. We demonstrate that the plasma membrane delivery of ZmPIP2;5, but not
the H+-ATPase PMA2, is reduced by dominant-negative Sp2
fragments of SYP121 from Arabidopsis and maize. This regulation is SYP121 specific, as Sp2 fragments of At-SYP71, AtSYP122, and At-SYP21 syntaxins had no effect on Zm-PIP2;5
traffic. We also show that the membrane osmotic water permeability coefficient of plant cells and oocytes coexpressing
Zm-PIP2;5 and SYP121-Sp2 is significantly reduced. However,
Figure 4. ZmSYP121-Sp2 Decreases the Pf of Xenopus Oocytes When
Coexpressed with Zm-PIP2;5.
(A) Pf of Xenopus oocytes (co)expressing Venus:ZmPIP2;5, Venus:
ZmPIP2;5 and 6His:ZmSYP121, Venus:ZmPIP2;5 and 6His:ZmSYP121Sp2, or 6His:ZmSYP121 and 6His:ZmSYP121-Sp2 or oocytes injected
with water (Mock). Each bar is the mean of 10 replicates. Error bars are
95% confidence intervals. Values were subjected to ANOVA and Tukey
posthoc analysis. Means that are significantly different (P < 0.05) are
indicated by different letters.
(B) Confocal images of oocytes (co)expressing Venus:ZmPIP2;5 (a),
Venus:ZmPIP2;5 and 6His:ZmSYP121 (b), (c) Venus:ZmPIP2;5 and 6His:
ZmSYP121-Sp2, Venus:ZmSYP121 (d), or Venus:ZmSYP121-Sp2 (e),
water-injected oocytes (f), or oocytes expressing Venus (g). The oocytes
were fixed in 4% (w/v) paraformaldehyde and analyzed by CLSM. Note
that all proteins mainly accumulated in the plasma membrane, except
Venus:ZmSYP121-Sp2, which showed a localization pattern similar to
that of Venus. Bars = 100 µm.
(C) Relative Venus fluorescence signal intensity in the plasma membrane
(M) compared with the cytoplasm (C) of oocytes expressing Venus:
ZmPIP2;5, Venus:ZmPIP2;5, and 6His:ZmSYP121, Venus:ZmPIP2;5 and
6His:ZmSYP121-Sp2, Venus:ZmSYP121, Venus:ZmSYP121-Sp2, or
Venus. Each bar is the mean of 20 replicates. Error bars are 95% confidence intervals. Values were subjected to ANOVA and Tukey posthoc
analysis. Means that are significantly different (P < 0.05) are indicated by
different letters.
(D) Oocyte total protein extracts were probed with antibodies raised
against AtSYP121. Protein extracts from oocytes expressing Venus:
ZmPIP2;5 and 6His:ZmSYP121-Sp2 (a), 6His:ZmSYP121-Sp2 (b), Venus:ZmPIP2;5 and 6His:ZmSYP121 (c), 6His:ZmSYP121 (d), Venus:
ZmSYP121 (e), or mock-injected sample (f).
(E) Oocyte total protein extracts were probed with antibodies raised
against Zm-PIP2;5. Protein extracts from oocytes expressing Venus:
ZmPIP2;5 (a), Venus:ZmPIP2;5 and 6His:ZmSYP121 (b), and Venus:
ZmPIP2;5 and 6His:ZmSYP121-Sp2 (c), or mock-injected sample (d). No
DTT was added to the samples, meaning that only dimers of Venus:
ZmPIP2;5 (;125 kD) are detected.
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The Plant Cell
in contrast with what is observed in plant cells, the Sp2 fragment did not affect the plasma membrane localization of ZmPIP2;5 in oocytes, suggesting a role of SYP121 in regulating
aquaporin activity in specific conditions. We now also demonstrate a direct interaction of maize SYP121 with Zm-PIP2;5.
These findings suggest an important role for SNAREs in the
regulation of the traffic and activity of plasma membrane
aquaporins.
The Syntaxin SYP121 Is a Specific Regulator of Zm-PIP2;5
Plasma Membrane Trafficking
Coexpression of mYFP:ZmPIP2;5 with the soluble ZmSYP121Sp2 fragment (cotransfected plasmid ratio of 1:1) leads to
a decrease in its plasma membrane abundance and, to an extent, its accumulation in intracellular structures. This intracellular
accumulation is further increased in cells cotransfected with the
plasmids encoding mYFP:ZmPIP2;5 and mCFP:ZmSYP121-Sp2
at a ratio of 1:3 (see Supplemental Figure 4 online). The total
amount of mYFP:ZmPIP2;5 fluorescence (plasma membrane and
internal signals) in these protoplasts was similar to the one found
in cells expressing mYFP:ZmPIP2;5 alone. These data are in accordance with the observation that the soluble SYP121-Sp2
fragment competes with the endogenous full-length SYP121 and
behaves as a dominant-negative (competitor) protein (Geelen
et al., 2002). From the time-lapse experiments, we found that
Figure 5. Zm-SYP121 and Zm-PIP2;5 Interact Both in Oocytes and in
Plant Cells.
(A) and (B) Copurification of solubilized proteins extracted from Xenopus
oocyte microsomal fractions on nickel–nitrilotriacetic acid resin. Elution
fractions were probed with antibodies raised against Zm-PIP2;5 in (A)
and against GFP in (B). Eluted fractions containing proteins extracted
from oocytes expressing 6His:ZmPIP2;5 (a), 6His:ZmPIP2;5 and Venus:
ZmSYP121 (b), and Venus:ZmSYP121 (c) or water-injected oocytes (d).
(C) BiFC in maize epidermal cells and mesophyll protoplasts. (a) Confocal images of maize epidermal cells transiently expressing mYFP:
ZmPIP2;5 and (b) stained with the plasma membrane stain, styryl dye
FM4-64. (c) Colocalization was determined as the relative fluorescence
intensities of mYFP and FM4-64 along the white dotted line. The black
dotted line above the spectra represents the white dotted lines shown in
(a) and (b). Note the matches between peaks for the two channels
corresponding to plasma membranes. (d) to (g) Maize epidermal cells
coexpressing mCFP:ZmSYP121 (d) and mYFP:ZmPIP2;5 (e). The
plasma membrane was stained with FM4-64 (f). Merged image of mCFP:
ZmSYP121, mYFP:ZmPIP2;5, and FM4-64 (g). (h) to (k) BiFC experiments in maize epidermal cells transfected by biolistic bombardment.
Confocal images of epidermal cells expressing VenusN:ZmPIP2;5 and
VenusC:ZmPIP1;2 (h), and VenusN:ZmPIP2;5 and VenusC:ZmSYP121 (i).
(j) Bright field. (k) YFP channel of cells expressing VenusN:PMA2 and
VenusC:ZmSYP121. (l) to (r) BiFC experiments in maize mesophyll protoplasts. Confocal images of protoplasts transiently coexpressing VenusN:ZmPIP2;5 and VenusC:ZmPIP1;2 ([l] and [m]) or VenusN:ZmPIP2;5
and VenusC:ZmSYP121 ([n] and [o]). (p) to (r) Protoplasts coexpressing
VenusN:PMA2 and VenusC:ZmSYP121. (p) Chlorophyll a autofluorescence. (q) YFP channel. (r) Bright field. Bars = 20 mm in (a) to (k)
and 5 mm in (l) to (r). a.u., arbitrary units.
(D) Emission spectra after excitation at 514 nm of mYFP:ZmPIP2;5 (black
solid line) and reconstituted Venus (gray dashed line). Fluorescent signal
was collected from 517 to 683 nm with an interval of 9.8 nm. mYFP and
reconstituted Venus share a similar emission pattern.
PIP Aquaporin Regulation by SNARE
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Table 1. FRET Efficiencies for the PIP/SYP121 Interaction Studies
Protein
FRET Efficiency 6
mCFP:ZmPIP1;2 + mYFP:ZmPIP2;5
mCFP:ZmSYP121 + mYFP:ZmPIP2;5
mCFP:ZmPIP2;5 + mYFP:ROP6
mCFP:ZmPIP1;2 + mYFP:ZmPIP2;5
mCFP:ZmSYP121 + mYFP:ZmPIP2;5
25.7
14.8
6.86
23.9
14.1
6
6
6
6
6
SD
(%)
9.56
3.12
9.26
5.42
7.96
No. of Measurements
Cell Type
69
35
20
35
50
Maize protoplasts
Maize protoplasts
Maize protoplasts
Tobacco epidermal cells
Tobacco epidermal cells
For each analysis, the average value of the FRET efficiency was determined as described in Methods.
mYFP:ZmPIP2;5 accumulates in the plasma membrane within 6 h
of transfection (see Supplemental Figure 3, Supplemental Movie
1, and Supplemental Movie Legend 1 online). However, in cells
coexpressing mYFP:ZmPIP2;5 and ZmSYP121-Sp2, the quantification of fluorescence intensity provides unequivocal evidence
that the traffic dynamics of mYFP:ZmPIP2;5 are affected primarily
at time points after the protein had reached the plasma membrane, suggesting an action of the Sp2 fragments on the vesicles
containing newly synthesized and recycling PIPs from the plasma
membrane. This is in agreement with the results obtained using
the vesicle-trafficking inhibitor brefeldin A, which prevents the
traffic from the Golgi apparatus to the plasma membrane and
inhibits the recycling of membrane protein to the plasma membrane (Geldner et al., 2001; Dhonukshe et al., 2007). Brefeldin A
blocks the plasma membrane trafficking of At-PIP2;1 in Arabidopsis roots (Paciorek et al., 2005) and phenocopies the KAT1 K+
channel trafficking phenotype in the syp121 mutant (Eisenach
et al., 2012). Indeed, SYP121 cycles actively between the TGN
and the plasma membrane (Reichardt et al., 2011). In addition, the
mCFP:ZmSYP121-Sp2 signal continuously rose during our experiments, demonstrating that the global protein synthesis is not
inhibited and did not constitute the origin of the decrease in mYFP:
ZmPIP2;5 plasma membrane fluorescence (see Supplemental
Figure 4 online).
The short-term FRAP kinetic experiments showed that only
20% 6 4.48% of YFP:ZmPIP2;5 is mobile at the cell periphery.
Similar results were obtained in Arabidopsis plants expressing
AtPIP2;1:GFP (Li et al., 2011; Sorieul et al., 2011; Luu et al., 2012).
Interestingly, coexpression with the dominant-negative SYP121Sp2 fragment reduced the mobile fraction to 7.02% 6 3.62% but
the fitted time constants were not affected (20.7 6 7.69 s and
19.64 6 6.58 s, respectively). We interpret this result as an inhibition of mYFP:ZmPIP2;5 recycling to the plasma membrane in
these cells. We did not detect any fluorescence loss in the
neighborhood of the bleached area, suggesting that little lateral
diffusion of ZmPIP2;5 occurred (see Supplemental Figure 4 online). While very low lateral membrane mobility was observed by
FRAP in normal conditions for several fluorescent tagged channels, including Arabidopsis KAT1, PIP2;1, and Nodulin 26-like
intrinsic protein NIP5;1, a boric acid channel, the mobility of AtKAT1 in the presence of the Sp2 fragment increased significantly,
indicating a role of the SYP121 in KAT1 distribution and behavior
at the membrane surface (Sutter et al., 2006b; Takano et al., 2010;
Sorieul et al., 2011; Luu et al., 2012). This was not observed for
Zm-PIP2;5, suggesting either that SYP121 is required for docking
this channel in the plasma membrane but not for its anchoring or
that in vivo interactions are more transient than they are for the K+
channel and therefore do not constitute the overriding factor
determining mobility.
The syntaxin SNAREs are well conserved among all eukaryotes (Yoshizawa et al., 2006). In plants, sequence comparison
allowed the identification of rice orthologs for most of the Arabidopsis Q-SNAREs (Sutter et al., 2006a). Our observation that
SYP121-Sp2 fragments from Arabidopsis and maize decreased
the plasma membrane trafficking of mYFP:ZmPIP2;5 in tobacco
epidermal cells and maize mesophyll protoplasts demonstrates
that the mechanism of SYP121-mediated plasma membrane
anchoring of PIP2 aquaporins is conserved between monocots
and dicots. In addition, the localization of the human AQP2 at the
apical membrane of epithelial cells of the collecting duct is also
specifically regulated by the syntaxin-3, SNARE-associated protein Snapin and SNAP33 complex (Procino et al., 2008; Mistry
et al., 2009), suggesting that the relative abundance of plasma
membrane aquaporins is regulated by SNARE complexes in all
eukaryotes.
The specificity of SYP121 in regulating plasma membrane
Zm-PIP2;5 trafficking is indicated by the fact that AtSYP122Sp2, AtSYP71-Sp2, and AtSYP21-Sp2 do not significantly alter the plasma membrane Zm-PIP2;5 abundance. In silico
search in the Gramene database (http://www.gramene.org/)
showed that At-SYP122, At-SYP71, and At-SYP21 have close
homologs in maize, indicating a possible conservation of the
trafficking regulation. Protein trafficking to the plasma membrane in Arabidopsis is defined by at least seven of the nine
Qa-SNAREs of the SYP1 subfamily (Grefen et al., 2011). AtSYP121 and its close homolog At-SYP122 localize to the
plasma membrane and their function is partially redundant
(Leyman et al., 2000; Collins et al., 2003; Uemura et al., 2004).
However, a coexpression study with a dominant-negative
mutant of Rab11, a GTPase required for plasma membrane
and cell plate trafficking, indicate that SYP121 and SYP122
drive two independent secretory events (Rehman et al., 2008).
Interestingly, similarly to what has been observed for AtSYP121-Sp2 in tobacco (Sutter et al., 2006b), ZmSYP121-Sp2
does not affect the plasma membrane delivery of the mCFP:
NpPMA2 H+ATPase in maize protoplasts.
SYP121 Affects the Membrane Osmotic Water Permeability
by Direct Interaction with Zm-PIP2;5
A first link between the trafficking and the regulation of ZmPIP2;5 activity by Zm-SYP121 derives from protoplast swelling
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Figure 6. PIP2 Proteins Interact with SYP121 in Mating-Based Spilt-Ubiquitin Assay.
(A) and (B) Mating-based split-ubiquitin assays were performed using At-PIP2;2 (A) and Zm-PIP2;5 (B) as baits, in each case with Zm-SYP121 and the
Arabidopsis ortholog At-SYP121 as the prey. Diploid yeast containing the water channels as bait and the SNAREs as prey or Nub-moiety peptides
(NubG = negative; NubI = positive control) were dropped in a dilution series (OD 1.0 and 0.1) onto the yeast synthetic media CSM-L-W- to verify mating
and on CSM-L-, W-, Ura-, H-, M-, and Ade- containing increasing Met levels to repress expression of the bait. Yeast growth was recorded after
incubation for 24 h (mating test) and for 48 h (interaction test) at 30°C.
(C) Expression of bait and prey fusion proteins was verified using anti-SYP121 antibody (Tyrrell et al., 2007) and VP16 (Abcam) antibodies. Molecular
masses of the primary bands in the protein gel blots are indicated below. Ponceau S staining (above) was recorded to monitor equal loading. The double
bands are characteristic of the SNAREs (Geelen et al., 2002).
PIP Aquaporin Regulation by SNARE
assays. A significant decrease in Pf was measured in protoplasts
coexpressing mYFP:ZmPIP2;5 and the ZmSYP121-Sp2 fragment. The most plausible hypothesis is that this Pf alteration results from a decrease in mYFP:ZmPIP2;5 abundance in the
plasma membrane. However, this interpretation does not preclude additional effects of ZmSYP121-Sp2 in directly modifying
the water channel activity of ZmPIP2;5. We observed that oocytes
coexpressing mYFP:ZmPIP2;5 and ZmSYP121-Sp2 were characterized by a lower Pf compared with oocytes expressing mYFP:
ZmPIP2;5 singly or in combination with Zm-SYP121. Significantly,
in these oocytes, no difference in the mYFP:ZmPIP2;5 expression
level and plasma membrane fluorescence intensity was detected,
suggesting that in this heterologous system, mYFP:ZmPIP2;5
trafficking could not explain the difference in activity.
In mammalian cells, the plasma membrane delivery and gating
of the cAMP-gated chloride channel and the inward rectifier
potassium channels (Kir) are regulated by syntaxin and other
exocytotic proteins (Naren et al., 1997; Leung et al., 2007). In
plants, At-SYP121 directly interacts with the KC1 K+ regulatory
subunit and modulates the activity of the AKT1/KC1 potassium
channel (Honsbein et al., 2009; Grefen et al., 2010). Similarly,
a direct interaction between Zm-PIP2;5 and Zm-SYP121 is
demonstrated by affinity chromatography copurification after
their expression in oocytes, yeast mating-based split-ubiquitin
assays, and microscopy-based techniques (BiFC and FRET) in
plant cells. These imaging approaches showed an interaction
between Zm-PIP2;5 and Zm-SYP121 in both the plasma
membrane and ER. This latter localization was predominant in
maize protoplasts. Considering that Zm-PIP2;5 and Zm-SYP121
use the secretory pathway to reach the plasma membrane, this
localization may simply reflect the lack of sufficient coordinate
partners necessary to correctly process and target the proteins
to the plasma membrane. Validation of in vivo interactions
comes from FRET measurements by acceptor photobleaching.
In our hands, this FRET methodology produces similar results to
the FRET measured by acceptor-sensitized emission. Interactions
between mYFP:ZmPIP2;5 and mCFP:ZmSYP121 are detected in
the plasma membrane, but the FRET efficiency was 10% weaker
than the efficiency measured in cells coexpressing mCFP:
ZmPIP1;2 and mYFP:ZmPIP2;5. Since no FRET was detected in
protoplasts coexpressing mCFP:ZmPIP2;5 and mYFP:ROP6,
a plasma membrane–localized GTPase, which does not interact
with Zm-PIP2;5 (Bischoff et al., 2000; Zelazny et al., 2007), we
concluded that FRET by acceptor photobleaching could be used
to investigate membrane protein interactions in tobacco epidermal
cells and maize protoplasts. Altogether, the microscopy-based
interaction data demonstrate that SYP121 physically interacts with
Zm-PIP2;5 in plant cells. These observations were further supported by yeast split-ubiquitin assays that demonstrated an
interaction of Zm-PIP2;5 with Zm-SYP121 or At-SYP121 in the
rescue of diploid yeast growth.
These data allow us to propose hypothetical models (Figure
7). In plant cells, the plasma membrane delivery of Zm-PIP2;5
during both constitutive recycling and anterograde transport
from the TGN vesicles is mediated by SYP121, either by a QaSNARE/VAMP vesicle recognition independent of Zm-PIP2;5
and/or by a direct docking interaction between Zm-PIP2;5,
SYP121, and VAMP (Figure 7A). In cells coexpressing Zm-
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PIP2;5 and ZmSYP121-Sp2, the truncated SNARE interacts
with Zm-PIP2;5 and VAMP, preventing the formation of the
SNARE complex with native Zm-SYP121 and, therefore, ZmPIP2;5 plasma membrane delivery (Figure 7B). In addition, experiments in Xenopus oocytes indicate that SYP121-Sp2 can
directly affect the activity of Zm-PIP2;5. In this system, the vesicular secretion of heterologously expressed protein appears
Figure 7. Hypothetical Models Summarizing the Regulation of ZmPIP2;5 Trafficking and Activity by Zm-SYP121.
(A) Regulation of Zm-PIP2;5 trafficking in plant cells coexpressing ZmPIP2;5 and full-length Zm-SYP121. Constitutive recycling and delivery of
newly synthesized Zm-PIP2;5 to the plasma membrane are regulated by
SYP121 through Qa-SNARE/VAMP and direct interactions. SYP121 allows a proper docking of Zm-PIP2;5 at the plasma membrane and regulates its amount.
(B) Regulation of Zm-PIP2;5 trafficking in plant cells coexpressing ZmPIP2;5 and the ZmSYP121-Sp2 fragment. (a) The dominant-negative
mutant SYP121-Sp2 might bind to Zm-PIP2;5 proteins, altering their
water channel activity and preventing their interaction with full-length
SYP121. (b) Constitutive recycling and delivery of newly synthesized
Zm-PIP2;5 to the plasma membrane are altered by the binding of
SYP121-Sp2 to VAMP and Zm-PIP2;5. This interaction does not allow
SNARE complex formation and the fusion of vesicles carrying Zm-PIP2;5
cargo to the plasma membrane. Additionally, SYP121-Sp2 might bind
resident ZmPIP2;5 proteins and inhibit their water transport activity. See
text for additional explanations.
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The Plant Cell
not to be specific (Mohun et al., 1981), explaining the fact that
SYP121-Sp2 does not affect Zm-PIP2;5 trafficking to the
plasma membrane. In cells (co)expressing Zm-PIP2;5 and ZmSYP121, the two partners interact at the plasma membrane and
possibly in the vesicle without affecting the trafficking or water
channel activity of Zm-PIP2;5. By contrast, in oocytes coexpressing Zm-PIP2;5 and ZmSYP121-Sp2, the plasma membrane delivery of ZmPIP2;5 is not affected, but its water channel
activity is significantly decreased. The observation that no significant increase in Pf was measured in maize protoplasts coexpressing mYFP:ZmPIP2;5 and mCFP:ZmSYP121-Sp2 compared
with mock cells, even if some mYFP fluorescence was detected in
the plasma membrane, suggest that, in plant cells, the Sp2 soluble fragment also inhibits Zm-PIP2;5 activity (Figure 7B). One
hypothesis is that the Sp2-soluble fragment interacts with ZmPIP2;5 cytosolic loops or extremities and modifies its conformation and gating (Törnroth-Horsefield et al., 2006). A modification of
AQP1-mediated water entry in pancreatic zymogen granules was
shown to be blocked after incubation with antibodies raised
against the C-terminal end of the protein (Cho et al., 2002). Because of steric constraint for accessibility, this kind of conformational change would not occur with membrane-anchored
SYP121, which would rather act to increase PIP stability in the
plasma membrane.
Is SYP121 a Stress-Responsive SNARE Involved
in the Maintenance of the Cell Turgor and Water
Balance in Plants?
It is striking to observe that the SNARE SYP121 regulates the
trafficking and activity of plasma membrane K+ channels and
aquaporins, transporters involved in the regulation of cell water
homeostasis. In addition, all of these components are regulated
by the stress hormone abscisic acid (Leyman et al., 1999;
Geelen et al., 2002; Parent et al., 2009). An attractive hypothesis
is that SYP121 acts as an essential component to coregulate ion
and water movement through the cell membrane.
Besides modulating trafficking and anchoring of KAT1,
SYP121 can directly modulate K+ channel activity. Split-ubiquitin,
BiFC, and coimmunoprecipitation experiments demonstrated that
SYP121, but not its closest homolog SYP122, directly interacts
with the KC1 regulatory subunit to form a tripartite SYP121-KC1AKT1 complex. Mutations in any of the three proteins selectively
suppressed the inward-rectifying K+ current in Arabidopsis root
epidermal protoplasts as well as K+ acquisition and growth in
seedlings when channel-mediated K + uptake was limiting
(Honsbein et al., 2009). Further characterization showed that
SYP121 can also interact with KAT1 (Grefen et al., 2010) and
that the FxRF motif located in the first 12 amino acid residues of
SYP121 was required for the selective interaction with the K+
channels (Grefen et al., 2010). These data indicate an essential
role of SYP121 in the connection between ion transport and
membrane traffic (Grefen et al., 2011; Honsbein et al., 2011). Our
results suggest that SYP121 could also regulate water homeostasis through modulation of aquaporin docking and possibly
activity. We showed here that SYP121 can physically interact
with Zm-PIP2;5 and acts as a regulator of its plasma membrane
delivery. The absence of direct modulation of Zm-PIP2;5 water
channel activity by full-length SYP121 led us to suggest that it
can rather play a role in coordinating water and K+ ion fluxes. We
propose that SYP121 acts to coordinate the plasma membrane
density of both PIP and K+ channels by regulating their membrane delivery and recycling. The next challenge will be to
monitor the dynamics of PIPs and K+ channels in the plasma
membrane of cells subjected to osmotic stress. However, as
SYP121 physically interacts with both PIPs and KC1 subunits,
we cannot exclude at this stage that SYP121 also plays a role as
a physical bridge between both proteins, resulting in the formation of a higher complex. It was suggested that PIPs could
act as turgor sensors in the plasma membrane that modulate the
conductance of K+ ion channels (Hill et al., 2004). The PIP/
SYP121/K+ channel tripartite regulation could coordinate the
turgor sensing with a tight tuning of ions and water movement
and content in growing cells, guard cells, or cells submitted to
drought stress. A direct link between ion transport, stomatal
aperture, and optimization of water use efficiency has recently
been established in the Arabidopsis syp121 loss-of-function
mutant growing in high light intensity and low relative humidity
(Eisenach et al., 2012). Importantly, we showed that protoplasts
isolated from syp121 leaves have a lower membrane water
permeability than do wild-type protoplasts, indicating a role of
SYP121 in aquaporin regulation. Altogether, our data add evidence to the concept of SNAREs being a molecular governor
(Grefen and Blatt, 2008; Honsbein et al., 2011) that coordinates
membrane transporter traffic and activity with the regulation of
water and solute transport in plant cells.
METHODS
Isolation and Transfection of Maize and Arabidopsis Protoplasts
Maize (Zea mays) B73 seedlings were grown, and mesophyll protoplast
isolation and transfection were performed as described previously (Zelazny et al., 2007). Arabidopsis thaliana Columbia-0 wild type and syp121
(syp121-1/pen1-1) mutants (Collins et al., 2003) were grown in soil under
250 mmol m22 s21 in long days (16:8 light [L]:dark [D]) at 22:21°C (L:D) with
a relative humidity of 70%. Mesophyll protoplasts were isolated as described previously (Ramahaleo et al., 1999).
Protoplast Swelling Assay
Maize and Arabidopsis protoplast swelling experiments were performed
as described previously (Moshelion et al., 2004; Volkov et al., 2007). The
solutions used for Arabidopsis protoplast swelling have been described
(Postaire et al., 2010). The selection of protoplasts (co)expressing mYFP:
ZmPIP2;5 and mCFP:ZmSYP121 or mCFP:ZmSYP121-Sp2 was achieved
due to their fluorescence emission.
Phylogenetic Analysis
The multiple alignment in Supplemental Figure 1 online was obtained
using the ClustalW algorithm with a Blosum62 scoring matrix, open gap
penalty of 10, and gap extension penalty of 0.5. The neighbor-joining
method was used to build the phylogenetic tree. Nodes’ significance was
inferred by 1000 bootstrap iterations.
Molecular Biology
PCR products encoding Np-PMA2, Zm-PIP2;5, Zm-SYP121, ZmSYP121Sp2, AtSYP121-Sp2, AtSYP122-Sp2, AtSYP21-Sp2, and AtSYP71-Sp2
PIP Aquaporin Regulation by SNARE
cDNAs were directionally subcloned using a uracil excision-based improved high-throughput USER cloning technique (Nour-Eldin et al., 2006)
into the USER-compatible plant expression vectors pCAMBIA2300 35Su
Nterm mYFP and pCAMBIA2300 35Su Nterm mCFP (Bienert et al., 2011).
This allowed the expression and in vivo visualization of the protein of
interest tagged with either mCFP or mYFP at the N terminus. For BiFC
experiments, new vectors were generated by adding a linker coding for
GSGGSGGS before the USER cassette in the pCAMBIA2300 35Su Nterm
Y155N and pCAMBIA2300 35Su Nterm Y155C vectors and after the
USER cassette in the pCAMBIA2300 35Su Cterm Y155N and pCAMBIA2300 35Su Nterm C155C vectors. This vector set was used to express
Zm-PIP2;5, Zm-PIP1;2, Zm-SYP121, and Np-PMA2 fused to the split
Venus. All cloning products were checked by sequencing. cDNAs encoding Zm-PIP2;5, Zm-SYP121, Zm-SYP121-Sp2, and Zm-PIP1;2 were
subcloned into the USER-compatible Xenopus laevis expression pNB1u,
pNBRGS-HISu, and pNB1YFPu vectors containing the YFP gene or
a sequence encoding a RGS-6 His tag 59 of the insertion site (Nour-Eldin
et al., 2006).
Particle Bombardment Transfection
Gold beads (0.6-µm diameter, 480 µg) were coated with 1 µg of plasmid
DNA. The median parts of the third leaf of 7-d-old etiolated maize
seedlings were placed in a Petri dish, which was positioned 3 cm below
the microprojectile stopping plate. Samples were bombarded once under
948 kPa partial vacuum at a rupture pressure of 1100 p.s.i. (7600 kPa)
using a PDS-1000/He Biolistic device (Bio-Rad). After transfection, leaf
explants were incubated overnight at 25°C in the dark on solid halfstrength Hoagland medium.
Confocal Microscopy
Detection of mCFP and mYFP fusion proteins was performed using
a Zeiss LSM710 confocal microscope equipped with a spectral detector.
Imaging of the maize mesophyll protoplasts was achieved with a PlanApochromat 363/1.40 oil immersion objective. mCFP and mYFP were
excited with the 445- and 514-nm laser lines, respectively. Emitted light
was collected through a dichroic mirror on detectors 450 to 510 nm
(mCFP) and 520 to 600 nm (mYFP). In each experiment, calibration of the
laser beam intensity, gain, and offset parameters were achieved on cells
expressing mYFP:ZmPIP2;5. The same parameters were then used in
image acquisitions performed on coexpressing cells, allowing a subsequent calculation of the plasma membrane fluorescence intensity. The
fluorescence intensity in the protoplast plasma membrane and intracellular compartments was quantified using an in-house developed
macro for the ImageJ software. First, the average thickness of the plasma
membrane was determined using the colocalized signals arising from
mYFP:ZmPIP2;5 and the steryl dye FM4-64 by averaging 100 measurements (10 measurements performed on 10 independent cells). The
mean value of the optical thickness of the protoplast plasma membrane
was 0.35 mm. Computation of the images was then performed in three
steps. First, the image was binarized, smoothed by the application of
a median filter, and, finally, converted into traces using the outline function
of the software. Second, the periphery trace was converted into the region
of interest (ROI-1), which thus contains the overall cell fluorescence. Third,
the ROI-1 was narrowed 0.35 mm in diameter to obtain ROI-2 that reflected the intracellular fluorescence. The fluorescence in the plasma
membrane is given by the difference between the integrated signal
densities (product of the surface area and the intensity of the signal)
measured in ROI-1 and ROI-2.
Time-lapse acquisitions were performed using a combination of the
time-lapse, tile scan, position, and Z-stack modules of the Zen 2009
software (Carl Zeiss MicroImaging). Image acquisition was done every 30
15 of 19
min for 16 h, using a C-Apochromat 340/1.20 water immersion objective
to maximize the size of the acquisition field.
FRAP experiments were performed as follows. Photobleaching (80
iterations, 514-nm laser beam at 100% [i.e., 7.5 mW]) was performed in
a defined ROI and recovery of fluorescence was monitored by acquisition
of one image every 5 s for 200 s. FRAP data were analyzed as previously
described (Sprague et al., 2004). The best fitting curve was obtained
according to a single exponential association using the “bottom to span”
algorithm in Prism software (GraphPad Software). The following equation
was used: y = span 3 (1 2 e-kt) + bottom, where the bottom is defined by
the minimal fluorescence intensity recorded after photobleaching, span is
the difference between the bottom and the calculated plateau, and k is the
binding constant defined by the half time of recovery, such as t1/2 = 0.69/k.
The BiFC assay was performed in tobacco (Nicotiana tabacum) epidermal cells transformed by Agrobacterium tumefaciens infiltration (Batoko
et al., 2000) or in polyethylene glycol–transfected maize protoplasts. In cells
exhibiting a fluorescence signal after excitation at 514 nm, an emission
spectrum was determined to validate the signal specificity. The subcellular
localization was assessed by Z-stack acquisition.
FRET was measured using either the acceptor photobleaching or
acceptor-sensitized emission methods. Acquisitions were performed on
two optical tracks, in sequential line mode. For the photobleaching
method, three images were acquired for both the mCFP and mYFP
channels, and then the mYFP was bleached by 80 iterations with fullpower laser illumination, while the fluorescence intensity of the mCFP was
simultaneously monitored along time. The FRET efficiency (E) was determined according the equation E = 12 IDA/ID, where IDA is the intensity of
the donor fluorescence before photobleaching and ID the fluorescence
intensity of the donor after photobleaching, as described elsewhere
(Kwaaitaal et al., 2010). The sensitized emission method was used on
a subpopulation of tobacco epidermal cells and protoplasts to confirm the
data obtained by acceptor photobleaching. One acquisition track was
composed of two acquisition channels to detect the mCFP and FRET
signals. mCFP was excited using the 445-nm laser beam, and the emitted
light was collected between 447 and 517 nm and between 523 nm and
630 nm for mCFP and FRET detection, respectively. The acceptor was
excited at 514 nm, and emitted light was collected between 523 and 628
nm. FRET efficiency was calculated according to the Youvan method
(Youvan et al., 1997) using the FRET module of Zen 2009 software (Carl
Zeiss MicroImaging).
Heterologous Expression in Xenopus Oocytes
In vitro cRNA synthesis, oocyte isolation, microinjection, and measurement of Pf and microsome isolation were performed as described by
Fetter et al. (2004). Oocytes were injected with 2 or 4 ng of cRNA encoding
the 6 His-tagged or YFP-tagged proteins, respectively. The cRNA was
quantified with a Nanodrop 1000 spectrophotometer (NanoDrop Technologies) and its integrity checked on an agarose gel. Oocytes expressing
the YFP-tagged constructs were fixed and sliced as described previously
(Sayers et al., 1997). Fluorescence was visualized by CLSM using a PlanNeofluar 310/0.30 objective as described above and quantified with
ImageJ software.
Mating-Based Split-Ubiquitin Assays
The haploid yeast strains THY.AP4 and THY.AP5 (Obrdlik et al., 2004;
Grefen et al., 2007) were transformed as described previously (Grefen
et al., 2009). Approximately 15 single colonies of bait (Cub-PLV) and prey
(Nub) constructs were selected following 3 d of growth on plates and
inoculated in vector-selective media for overnight growth. Liquid cultures
were harvested at an OD600 of 2 to 3 and resuspended in yeast-extract
peptone dextrose medium. Equal volumes (20 µL) of each bait and prey
16 of 19
The Plant Cell
were mixed and dropped on YPD plates. After 6 to 8 h incubation at 30°C,
yeast was scraped off and streaked on vector-selective media (CSM-L-,
W-, and Ura-). After ;1 to 2 d of growth, the diploid yeast was used to
inoculate liquid vector-selective media and grown overnight. The next
day, serial dilutions at OD600 1.0 and 0.1 in water were dropped, 7 mL per
spot, onto plates without and with the addition of 5, 50, and 500 µM Met
on interaction-selective media (CSM-L-, W-, Ura-, Ade-, H-, and M-).
Growth was monitored after 2 or 3 d at 30°C. Control plates on vectorselective media were incubated for 24 h only to verify that an equal
amount of yeast had been dropped. To verify expression, haploid yeast
was harvested prior to mating and analyzed via immunoblotting using
polyclonal antibodies against SYP121 and VP16, as described previously
(Grefen et al., 2009; Honsbein et al., 2009).
Solubilization and Purification of His:ZmPIP2;5
and Immunodetection
Microsomes were resuspended in 13 PBS buffer. Three hundred micrograms of proteins were solubilized with 1% (w/v) octyl-b-D-glucopyranoside in a final volume of 400 mL and incubated for 2 h on a rotary
wheel at room temperature. Unsolubilized material was removed by
centrifugation at 169,000g for 20 min, and the supernatant was added to
100 mL of Ni2+-nitriloacetic acid agarose matrix (Qiagen) preequilibrated
with 13 PBS buffer containing 1% [w/v] octyl-b-D-glucopyranoside and
10 mM imidazole. The mixture was incubated for 2 h on a rotary wheel at
room temperature, loaded on a column, and washed three times with 1 mL
of 13 PBS buffer containing 10 mM imidazole and 0.05% Tween 20.
Bound proteins were eluted with 100 mL Laemmli buffer. Proteins (50 µL/
well) were electrophoresed by SDS-PAGE, transferred to a nitrocellulose
membrane, and immunodetected with antibodies directed against GFP
(Duby et al., 2001), SYP121 (Tyrrell et al., 2007), and Zm-PIP2;5 (Hachez
et al., 2006) by the enhanced bioluminescence method.
Supplemental Figure 5. Fluorescence Recovery after Photobleaching.
Supplemental Figure 6. Pf Values Measured after 15, 30, and 45 s of
Protoplast Swelling.
Supplemental Figure 7. Zm-SYP121 and Zm-PIP2;5 Interact in
Tobacco Epidermal Cells.
Supplemental Figure 8. FRET Experiments.
Supplemental Figure 9. Comparison of Sensitized Emission and
Acceptor Photobleaching FRET Efficiencies.
Supplemental Data Set 1. Multiple Alignments of the Full-Length
Protein Sequences of Qa SNARE Used to Build the Phylogenetic Tree
of Supplemental Figure 2 Online.
Supplemental Movie 1. Movie of Time-Lapse Acquisition of mCFP:
ZmSYP121-Sp2 and mYFP:ZmPIP2;5 Accumulation in Maize Mesophyll Protoplasts.
Supplemental Movie Legend 1. Legend for Supplemental Movie 1.
ACKNOWLEDGMENTS
We thank the imaging platform IMABIOL. This work was supported by
grants from the Belgian National Fund for Scientific Research (FNRS), the
Interuniversity Attraction Poles Programme–Belgian Science Policy, the
“Communauté française de Belgique–Actions de Recherches Concertées”, and by grants BB/F001630/1 and BB/H0009817/1 to M.R.B. from
the UK Biotechnology and Biological Sciences Research Council. A.B.
was a postdoctoral research fellow at the FNRS. G.P.B. was supported
by an individual Marie Curie European fellowship and by the FNRS. A.S.C.
was a research fellow at the “Fonds pour la Formation à la Recherche dans
l’Industrie et dans l’Agriculture.”
Accession Numbers
AUTHOR CONTRIBUTIONS
Sequence data from this article can be found in the GenBank/EMBL and
maize sequencing project data libraries (http://www.maizesequence.org/
index.html; Schnable et al., 2009) under the following accession numbers:
At-SYP111, NP_172332; At-SYP112, NP_179418.2; At-SYP121,
NP_187788; At-SYP122, NP_190808; At-SYP123, NP_192242; AtSYP124, NP_176324; At-SYP125, NP_172591; At-SYP131, NP_187030;
At-SYP132, NP_568187; Os-SYP111, ABB22783; Os-SYP112, ABA96047;
Os-SYP121, ABF99241; Os-SYP124, NP_001172852; Os-SYP131,
NP_001058959; Os-SYP132, NP_001056925; Zm-SYP111, NP_001149999;
Zm-SYP112, GRMZM2G168017; Zm-SYP121, NP_001150776, ZmSYP124, GRMZM2G411561; Zm-SYP123, GRMZM2G416733; ZmSYP132, NP_001149376; Zm-SYP131, GRMZM2G330772; Np-PMA2,
AAA34052; and Zm-PIP2;5, AF130975.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Unrooted Phylogenetic Tree of the Plasma
Membrane–Localized Qa SNAREs.
Supplemental Figure 2. Intracellular Accumulation of mYFP:
ZmPIP2;5 in Protoplasts Transfected with the Plasmids Encoding
mCFP:ZmSYP121-Sp2 and mYFP:ZmPIP2;5 at a 3:1 Ratio.
Supplemental Figure 3. Still Images of Supplemental Movie 1,
Showing Time-Lapse Acquisition of mCFP:ZmSYP121-Sp2 and
mYFP:ZmPIP2;5 Accumulation in Maize Mesophyll Protoplasts.
Supplemental Figure 4. ZmSYP121-Sp2 Affects Plasma Membrane
Delivery but Not Synthesis of mYFP:ZmPIP2;5.
A.B. designed and performed research, analyzed data, developed new
analytic tools, and wrote the article. E.B. performed research and
analyzed data. G.P.B. designed the research, provided molecular tools,
and analyzed data. A.S.C. provided molecular tools, performed research,
and analyzed the data. A.E. provided technical assistance. C.G.
performed research, analyzed data, and wrote the article. F.C. designed
the research and, together with M.R.B., analyzed data and wrote the
article.
Received June 19, 2012; revised July 23, 2012; accepted August 1,
2012; published August 31, 2012.
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Selective Regulation of Maize Plasma Membrane Aquaporin Trafficking and Activity by the
SNARE SYP121
Arnaud Besserer, Emeline Burnotte, Gerd Patrick Bienert, Adrien S. Chevalier, Abdelmounaim
Errachid, Christopher Grefen, Michael R. Blatt and François Chaumont
Plant Cell; originally published online August 31, 2012;
DOI 10.1105/tpc.112.101758
This information is current as of June 15, 2017
Supplemental Data
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