This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been
edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online
reduces the time to publication by several weeks.
Phosphatidylinositol 4,5-Bisphosphate Influences PIN
Polarization by Controlling Clathrin-Mediated Membrane
Trafficking in Arabidopsis
CW
Till Ischebeck,a,1 Stephanie Werner,a,b,1,2 Praveen Krishnamoorthy,c Jennifer Lerche,a,b Mónica Meijón,d
Irene Stenzel,a,b Christian Löfke,e,3 Theresa Wiessner,f Yang Ju Im,g Imara Y. Perera,g Tim Iven,a Ivo Feussner,a
Wolfgang Busch,d Wendy F. Boss,g Thomas Teichmann,e Bettina Hause,f Staffan Persson,c and Ingo Heilmanna,b,4
a Department
of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University Göttingen, 37077
Goettingen, Germany
b Department of Cellular Biochemistry, Institute for Biochemistry, Martin-Luther-University Halle-Wittenberg, 06120 Halle (Saale),
Germany
c Max-Planck-Institute for Molecular Plant Physiology, D-14476 Potsdam-Golm, Germany
d Gregor-Mendel-Institute for Molecular Plant Biology, 1030 Vienna, Austria
e Department of Plant Cell Biology, Albrecht-von-Haller-Institute for Plant Sciences, Schwann-Schleiden Centre, Georg-AugustUniversity Göttingen, 37077 Goettingen, Germany
f Department of Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), Germany
g Department of Plant Biology, North Carolina State University, Raleigh, North Carolina 27695
The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during vegetative plant growth
remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and
PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P2
levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double
mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux
carriers PIN-FORMED (PIN1)–green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution.
Furthermore, fewer brefeldin A–induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2
mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxindependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and
polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns
in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P2 in the regulation of clathrin-mediated endocytosis.
Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and
pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for
PtdIns(4,5)P2 in the control of clathrin dynamics and, in auxin distribution in Arabidopsis.
INTRODUCTION
An important example of polarity in vegetative plant tissues is
the formation and maintenance of a stable auxin gradient in the
root tip, which is required for root development and gravitropism
(Dhonukshe et al., 2008; Bennett and Scheres, 2010). The
1 These
authors contributed equally to this work.
address: Gregor-Mendel-Institute for Molecular Plant Biology,
Dr. Bohr-Gasse 3, 1030 Vienna, Austria.
3 Current address: Institute for Applied Genetics and Cell Biology,
Muthgasse 18, 1190 Vienna, Austria.
4 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: Ingo Heilmann (ingo.
[email protected]).
C
Some figures in this article are displayed in color online but in black and
white in the print edition.
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.113.116582
2 Current
directional transport of auxin in the root is mediated by auxin efflux
carrier proteins of the PIN-FORMED (PIN) family, membrane integral transporters that exhibit asymmetric plasma membrane localization in root cells (Blilou et al., 2005; Tanaka et al., 2006).
Directional auxin transport and the perpetuation of a stable auxin
gradient in the root require concerted action of different PIN isoforms (Petersson et al., 2009). On the cellular level, the polar
subcellular distribution of PIN proteins to the basal or apical
plasma membrane of a cell defines the direction of auxin transport
(Blilou et al., 2005; Tanaka et al., 2006). This important asymmetric
distribution of plasma membrane associated PIN proteins is sustained by constant recycling and predominant delivery of the
proteins to certain areas of the plasma membrane (Boutté et al.,
2006; Kleine-Vehn et al., 2008). Thus, the polarization of PIN
proteins involves internalization of the proteins from the membrane, which depends on recruitment to clathrin-coated vesicles
(CCVs; Dhonukshe et al., 2007; Kitakura et al., 2011).
Various findings suggest that the minor regulatory phospholipid
phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] (Balla, 2006;
The Plant Cell Preview, www.aspb.org ã 2013 American Society of Plant Biologists. All rights reserved.
1 of 18
2 of 18
The Plant Cell
Thole and Nielsen, 2008; Heilmann, 2009) contributes to the
subcellular polarization of PIN proteins and, thus, to directional
auxin transport. For instance, auxin-dependent gravitropic curvature correlated with the production of PtdIns(4,5)P2 in maize
vesicle trafficking in polar growing cells (Ischebeck et al., 2008,
2010a; Kusano et al., 2008; Sousa et al., 2008; Stenzel et al.,
2008). Furthermore, PtdIns(4,5)P2 has been proposed to influence clathrin-mediated endocytosis in plants (König et al.,
2008b; Zhao et al., 2010). Finally, PIN cycling is perturbed in root
tips of an Arabidopsis thaliana pip5k2 mutant (Mei et al., 2012).
However, despite these multiple lines of evidence, the mechanism behind these observations has remained obscure.
In Arabidopsis, PtdIns(4,5)P2 is formed by an enzyme family of
11 phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases)
(Mueller-Roeber and Pical, 2002). Arabidopsis PI4P 5-kinases
often occur as pairs of closely related sister enzymes (Stenzel
et al., 2012), and examination of single and double T-DNA
insertion mutants indicates that the sister isoenzymes can
display redundant functions (Ischebeck et al., 2008; Sousa
et al., 2008; Ischebeck et al., 2011). Here, we eliminated two
ubiquitously expressed PI4P 5-kinases, PIP5K1, an active PI4P
5-kinase (Mikami et al., 1998) expressed in all plant organs with
predominant expression in procambial cells (Elge et al., 2001),
and its closely related isoform PIP5K2 (Stenzel et al., 2008;
Camacho et al., 2009; Mei et al., 2012). We show that the pip5k1
pip5k2 double mutant displays altered auxin transport and
perturbed PIN1-GFP (for green fluorescent protein) and PIN2GFP recycling. Our data indicate that polarization of PIN proteins requires PtdIns(4,5)P2, which influences the formation of
clathrin foci at the plasma membrane and possibly affects the
internalization of clathrin coated vesicles.
RESULTS
The PI4P 5-Kinases PIP5K1 and PIP5K2 Are Ubiquitously
Expressed in Arabidopsis
To examine the role of PI4P 5-kinases, we first identified
Arabidopsis genes encoding PI4P 5-kinase isoforms with potential roles in vegetative tissues using transcript array information
accessible through Genevestigator (Zimmermann et al., 2004). In
previous experiments, recombinant PIP5K1 and PIP5K2 displayed the highest specific activities among ubiquitously expressed PI4P 5-kinases from Arabidopsis (Stenzel et al., 2008);
therefore, we chose these genes for further characterization. The
PIP5K1 and PIP5K2 sequences cluster together on a subclade
of a phylogenetic tree of all deduced Arabidopsis PI4P 5-kinase
amino acid sequences (Stenzel et al., 2012), suggesting related
functionality. The third gene in this subclade, PIP5K3, is restricted in expression to roots and affects root hair formation
(Kusano et al., 2008; Stenzel et al., 2008). All other PI4P 5-kinase
isoforms are more distantly related and notably different from
PIP5K1 and PIP5K2. We verified expression of PIP5K1 and
PIP5K2 in vegetative tissues using 1500-bp promoter fragments
of the genes to drive the expression of a b-glucuronidase (GUS)
reporter construct in transgenic plants, followed by histochemical
staining, and independently by quantitative real-time RT-PCR
(see Supplemental Figure 1 online). The promoter-GUS experiments indicated that PIP5K1 and PIP5K2 are expressed already
early in development (see Supplemental Figures 1A and 1B
online) and have similar expression patterns in vegetative tissues (see Supplemental Figures 1C to 1J online) as well as in
flowers and pollen (see Supplemental Figures 1K to 1N online).
Promoter activity was especially high in procambial tissues (see
Supplemental Figures 1G and 1H online) and the root tips (see
Supplemental Figures 1A, 1B, 1I, and 1J online), as previously
noted for PIP5K1 (Elge et al., 2001). Quantitative real-time
RT-PCR analysis supports expression of both PIP5K1 and PIP5K2
in leaves, flowers, roots, whole seedlings, and flower buds (see
Supplemental Figure 1O online).
A pip5k1 pip5k2 Double Mutant Exhibits Reduced
PtdIns(4,5)P2 and Severely Impaired Growth
We next isolated homozygous Arabidopsis T-DNA mutant lines
with exon insertions in the genes PIP5K1 (SALK_146728) or
PIP5K2 (SALK_012487) (Figure 1A). We inferred homozygosity
from the inability to amplify wild-type alleles by PCR, concomitant with positive amplification of T-DNA–tagged alleles. T-DNA
insertions were found in the first exon of pip5k1, 657 bp
downstream from the ATG, and between the first exon and the
first intron of pip5k2, 1185 bp downstream from the ATG. A
pip5k1 pip5k2 double mutant was obtained by crossing homozygous pip5k1and pip5k2 mutant lines, and the combined
genotype was verified by PCR. Quantitative real-time RT-PCR
showed that transcripts of PIP5K1 or PIP5K2 were reduced
below the limits of detection in the respective single mutants
(Figure 1B). Interestingly, reduction of PIP5K1 or PIP5K2 transcripts was accompanied by elevated levels of the transcripts of
the other PI4P 5-kinase gene over the level of wild-type controls
(Figure 1B). This indicates a compensatory effect at the level of
transcription and complicates the interpretation of single mutant
phenotypes. By contrast, the pip5k1 pip5k2 double mutant showed
reduced levels of both transcripts (Figure 1B).
We measured the levels of PtdIns(4,5)P2 and various other
phospholipids for wild-type controls, the two single mutants, the
pip5k1 pip5k2 double mutant, and for a double mutant line
ectopically expressing PIP5K1:EYFP (for enhanced yellow fluorescent protein) under an intrinsic 1500-bp promoter fragment
by quantification of unlabeled compounds (Figure 1C; see
Supplemental Figure 2 online). Additionally, PtdIns(4,5)P2 was
independently quantified also by radiolabeling using [32P]Pi of
wild-type controls, the two single mutants, and the pip5k1
pip5k2 double mutant (Figure 1D). The most prominent changes
in lipid levels were observed for PtdIns(4,5)P2, which was reduced in the double mutant by 44 and 32% of wild-type levels
according to nonlabeled analysis (Figure 1C) and radiolabeling
(Figure 1D), respectively. The single mutants showed a less
pronounced reduction of PtdIns(4,5)P2. The reduction of
PtdIns(4,5)P2 in the pip5k1 pip5k2 double mutant was accompanied by increases in phosphatidylinositol 4-phosphate and phosphatidylinositol, its biosynthetic precursors (Figure 1C). Among
structural phospholipids, the level of phosphatidylcholine was
slightly decreased (see Supplemental Figure 2 online), possibly
indicating adaptive processes of membranes to a reduction in
PtdIns(4,5)P2 Controls PIN Cycling
3 of 18
PtdIns(4,5)P2. Other lipids tested, including phosphatidylethanolamine and phosphatidic acid, did not display notably altered
abundance in the single or double mutants (see Supplemental
Figure 2 online).
Single homozygous pip5k1 or pip5k2 mutants showed only
mildly altered growth (Figure 2A). By contrast, the pip5k1 pip5k2
double mutant displayed a strong growth defect (Figure 2A)
characterized by reduced leaf expansion (Figures 2A and 2C),
reduced root elongation in the light (Figures 2B and 2C), and
a reduction in apical dominance resulting in multiple (up to 50)
shoots from a single plant at a very late stage of development
(Figure 2D). Senescence was delayed in the double mutant and
plants could reach an age of over 10 months in long-day conditions. The pip5k1 pip5k2 double mutants did not make seeds.
As the double mutant did not produce flowers or siliques, double
mutants were propagated as segregating offspring of selfed
plants genotyped as pip5k1(2/2) pip5k2(+/2). Plants homozygous
for pip5k1 or pip5k2 and heterozygous for the respective other
allele [genotypes pip5k1(2/2) pip5k2(+/2) or pip5k1(+/2) pip5k2(2/2)]
displayed phenotypes intermediate between those of the single
mutants and the homozygous double mutant (see Supplemental
Figure 3 online). Ectopic expression of either untagged PIP5K1 or
PIP5K2 (data not shown) or PIP5K1:EYFP or PIP5K2:EYFP under
the native 1500-bp promoter fragments in the pip5k1 pip5k2
double mutant produced phenotypes similar to the single mutants
or even the wild-type controls. This observation supports the notion
that the fluorescence-tagged variants of PIP5K1 and PIP5K2
retained full functionality in vivo.
Gravitropic Bending and Auxin Distribution Are Impaired
in the pip5k1 pip5k2 Double Mutant
The phenotype of the pip5k1 pip5k2 double mutant resembles
that of plants deficient in biosynthesis or perception of auxin
(Palme and Gälweiler, 1999). To better characterize whether
auxin-dependent responses were altered in plants with reduced
Figure 1. Characterization of T-DNA Insertion Mutants Lacking the
Closely Related PI4P 5-Kinase Isoforms PIP5K1 and/or PIP5K2.
T-DNA insertion lines for PIP5K1 (SALK_146728) and PIP5K2
(SALK_012487) were isolated and tested for the abundance of relevant
transcripts and lipids.
(A) Graphic representation of the position of the insertions. The T-DNA
insertion in pip5k1 is located in the first exon and that in pip5k2 at the
transition of the first intron to the second exon. Black boxes indicate
exons; lines between boxes indicate introns. Arrows indicate the approximate positions of PCR primers used for genotyping and for transcript detection in cDNA preparations.
(B) Changes in transcript levels for PIP5K1 or PIP5K2 according to
quantitative real-time RT-PCR analysis, as indicated. Data represent the
means of three independent experiments 6 SD. Asterisks represent a
significant difference in transcript abundance compared with wild-type
controls according to a two-tailed Student’s t test (**P < 0.01). CT, cycle
threshold.
(C) Levels of phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate
(PtdIns4P), and PtdIns(4,5)P2 in wild-type controls, the pip5k1 and pip5k2
single mutants, the pip5k1 pip5k2 double mutant, and a double mutant
line ectopically expressing PIP5K1 under an intrinsic promoter fragment
(compl.), according to nonlabeled mass determination. Ten-day-old
seedlings were grown on MS agar plates and frozen in liquid nitrogen.
Lipids were extracted and separated by thin layer chromatography, and
the levels of PtdIns(4,5)P2 were determined by quantifying the associated
fatty acids by gas chromatography and mass spectrometry. Data represent the means of three independent experiments 6 SD. Asterisks indicate significant differences from the wild type according to a Student’s
t test (*P < 0.05; **P > 0.01).
(D) Determination of PtdIns(4,5)P2 levels in wild-type controls, the pip5k1
and pip5k2 single mutants, and the pip5k1 pip5k2 double mutant according to radiolabeling with [32P]Pi. Twelve-day-old Arabidopsis seedlings grown on MS agar plates were incubated for 30 min in liquid MS
medium containing [32P]Pi. Lipids were extracted and separated by thin
layer chromatography, and the radioactivity on the thin layer chromatography plates determined by liquid scintillation counting. The amount
of [32P]-labeled PtdIns(4,5)P2 was calculated as the percentage of the
total [32P] incorporated in the phospholipids of each sample. Data represent the means of three independent experiments 6 SD. Asterisks indicate significant differences from the wild type according to a Student’s
t test (*P < 0.05; **P < 0.01).
4 of 18
The Plant Cell
Figure 2. pip5k1 pip5k2 Double Mutant Plants Display Severe Growth Defects.
Arabidopsis wild-type controls, single and double mutant plants, and double mutant plants ectopically expressing PIP5K1-EYFP or PIP5K2-EYFP were
grown on soil or on MS media, and their phenotype was documented.
(A) Plants grown in soil under 16 h of light/8 h of darkness in the greenhouse for 5 weeks. Inset, identical plants after 4 weeks. Genotypes as indicated.
compl. 1, compl. 2, controls ectopically expressing PIP5K1-EYFP or PIP5K2-EYFP under their respective intrinsic promoters in the pip5k1 pip5k2
background.
(B) Seedlings grown for 10 d on MS medium under 24 h of light on a vertical plate. Left, wild-type controls; right, pip5k1 pip5k2 double mutants.
(C) Root length and leaf area were measured from seedlings grown on MS medium under 24 h of light after 7 d (gray bars) or 14 d (black bars). For
determination of root length, plants were grown on vertical plates. Data represent mean values 6 SE of at least 20 seedlings. Double asterisks indicate
significant differences from the wild type according to a Student’s t test (**P < 0.01).
(D) Plants were grown under 16 h of light/8 h of darkness in the greenhouse for 10 months. Double mutant plants showed a delay in senescence and
a loss of apical dominance. The image is representative for 10 plants observed over the prolonged growth period.
[See online article for color version of this figure.]
PtdIns(4,5)P2 Controls PIN Cycling
expression of PIP5K1 and/or PIP5K2, we monitored gravitropic
bending of roots and hypocotyls. Seedlings were grown in the
dark for 7 d on vertical agar plates, the plates were rotated by
135° for 24 h as previously described (Fukaki et al., 1996), and
gravitropic curvature was recorded (Figure 3). Compared with
wild-type controls, gravitropic curvature of hypocotyls and roots
was slightly reduced in pip5k1 or pip5k2 single mutants and
substantially reduced in the pip5k1 pip5k2 double mutant (Figure 3). A complemented double mutant line ectopically expressing pPIP5K1:PIP5K1-EYFP displayed curvature of roots
and hypocotyls similar to wild-type controls (Figure 3). The
elongation of dark-grown roots (see Supplemental Figure 4A
online) and hypocotyls (see Supplemental Figure 4B online) was
determined for all lines tested and not found to be significantly
different.
Because the phenotype of the pip5k1 pip5k2 double mutant
included reduced root growth, apical dominance, and gravitropic bending, we speculated that these aspects might be
a consequence of defects in auxin signaling. The wild-type
controls and the double mutant showed similar levels of endogenous auxin (Figure 4A). By contrast, the pip5k1 pip5k2
5 of 18
double mutant showed significantly reduced basipetal auxin
transport (Figure 4B). Auxin distribution was also tested by monitoring DR5-driven GFP distribution in roots (see Supplemental
Figure 5A online). In wild-type controls the DR5-dependent GFP
distribution reflected the characteristic auxin maximum in the
root tip (see Supplemental Figure 5A online, top left), and the
signal was further intensified by the exogenous application of
indole 3-acetic acid (IAA; see Supplemental Figure 5A online,
top right). By contrast, no GFP fluorescence was observed in
pip5k1 pip5k2 double mutants, neither in the absence nor the
presence of exogenous IAA (see Supplemental Figure 5A online,
bottom panels). The presence of the DR5:GFP construct was
confirmed by PCR. To rule out genetic effects of incrossing, an
independent DR5:GUS reporter line was created in the pip5k1
pip5k2 background (Figure 4C) and analyzed for responses to
endogenous and exogenous auxins. As for the DR5:GFP reporter, the DR5:GUS reporter also did not indicate an auxin
maximum in the pip5k1 pip5k2 double mutant. Reporter activity
only marginally increased in response to exogenous auxins,
such as a-naphthalene acetic acid or 2,4-dichlorophenoxyacetic
acid (2,4-D) (Figure 4C). Importantly, the spatial distribution of
Figure 3. Altered Gravitropic Response in pip5k1 and pip5k2 Single Mutants and the pip5k1 pip5k2 Double Mutant.
Gravitropic curvature and root growth was tested in wild -type controls, pip5k1 and pip5k2 single mutants, the pip5k1 pip5k2 double mutant, and plants
controls ectopically expressing PIP5K1-EYFP (compl. 1). Bending angles of hypocotyls and roots were recorded 24 h after rotation of 135° and scored
in categories of 30°. The length of the bars indicates the percentage of plants in each category. Wild type, data from 242 plants; pip5k1, data from 214
plants; pip5k2, data from 329 plants; pip5k1 pip5k2 double mutant, data from 288 plants; compl. 1, data from 198 plants.
[See online article for color version of this figure.]
6 of 18
The Plant Cell
the DR5-driven GUS signal in the pip5k1 pip5k2 double mutant
differed substantially from that in wild-type controls (Figure 4C).
To test whether pip5k1 pip5k2 mutants were still sensitive to
exogenous auxins, we treated wild-type controls and double
mutants with auxin and monitored root elongation over a period
of 6 d (see Supplemental Figure 5B online). The double mutant
displayed substantially reduced root growth (compared with
Figures 2B and 2C), within the limits of mutant growth,
a-naphthalene acetic acid, 2,4-D, and IAA had similar inhibitory effects on root elongation as in wild-type controls (see Supplemental
Figure 5B online). Although the DR5 reporter does not provide
bona fide information on auxin levels, the data suggested that
auxin perception was not impaired in the pip5k1 pip5k2 double
mutant. Furthermore, this experiment demonstrated that the application of exogenous auxins did not aid root growth in the double
mutant. Overall, the data presented in Figure 4 and Supplemental
Figure 5 online indicate that reduced levels of PtdIns(4,5)P2 parallel
reduced capability to maintain basipetal auxin transport in
root tips.
Overexpression of PIP5K1 and PIP5K2 Results
in Auxin-Related Growth Defects
Figure 4. Auxin Distribution Is Impaired in the pip5k1 pip5k2 Double
Mutant.
Levels and distribution of auxin were analyzed in wild-type controls and
in the pip5k1 pip5k2 double mutant.
(A) Levels of free IAA in 2-week-old seedlings according to HPLC–mass
spectrometry analysis. Data represent the mean 6 SE of two independent
experiments, each with three independent biological replicates. A Student’s
t test indicates no significant difference between controls and the double
mutant (P < 0.23).
(B) Relative basipetal auxin transport was determined in 10-d-old wildtype controls and in 14-d-old pip5k1 pip5k2 seedlings with similar root
lengths grown on vertical plates. Data represent the mean 6 SD from
three independent experiments. The asterisk indicates a significant difference from the control according to a Student’s t test (P < 0.05).
(C) Auxin distribution in the wild type (top) and pip5k1 pip5k2 double
mutant (bottom) according to DR5:GUS expression. Patterns after application of exogenous auxins, as indicated. The results are representative
for three independent treatments. 2,4-D, 2,4-dichlorophenoxyacetic acidNAA,
a-naphthalene acetic acid.
[See online article for color version of this figure.]
To examine the biochemical functionality of PIP5K1 and PIP5K2
variants used in this study, we recombinantly expressed the
proteins as fusions to N-terminal maltose binding protein tags
used to enhance solubility (see Supplemental Figure 6A online).
Biochemical characterization showed that EYFP-tagged variants of PIP5K1 and PIP5K2 displayed catalytic activity in vitro
(see Supplemental Figure 6B online). In vitro catalytic activity did
not differ between enzyme variants carrying only an N-terminal
maltose binding protein tag or an additional C-terminal EYFP tag
(see Supplemental Figure 6B online), consistent with previous
reports (Ischebeck et al., 2008, 2011; Stenzel et al., 2008, 2012).
Heterologous expression of untagged or fluorescence-tagged
variants of either PIP5K1 or PIP5K2 restored the ability to grow
at prohibitive temperature in a Saccharomyces cerevisiae strain
carrying an allele of MSS4 encoding a temperature-sensitive
variant of the PI4P 5-kinase MSS4p (Homma et al., 1998) (see
Supplemental Figure 6C online). The data confirm catalytic activity of PIP5K1 and PIP5K2 and indicate that fluorescencetagged variants of PIP5K1 and PIP5K2 were functional in vitro,
corroborating the mutant complementation tests (compared with
Figure 2).
To investigate the subcellular distributions of the proteins and
to assess the impact of overproduction of the two kinases,
we overexpressed fluorescence-tagged versions of PIP5K1 and
PIP5K2 in Arabidopsis. As the fluorescently tagged PIP5K1 and
PIP5K2 under their own promoters displayed only very weak
fluorescence, we replaced the native promoters with estradiolinducible promoters of the pMDC7 system (Curtis and Grossniklaus,
2003), which should induce gene expression strongly upon
estradiol treatment. We verified induction of the transgenes by
monitoring EYFP fluorescence (Figure 5A). In root tip cells of
these lines, EYFP-PIP5K1 and PIP5K2-EYFP displayed clear
plasma membrane localization and weak diffuse cytosolic fluorescence (Figure 5A), characteristic of soluble enzymes that
associate peripherally with the plasma membrane, and in
PtdIns(4,5)P2 Controls PIN Cycling
particular resembling localization patterns previously reported
for other plant PI4P 5-kinases in other cell types (Ischebeck
et al., 2008, 2010b, 2011; Kusano et al., 2008; Sousa et al.,
2008; Stenzel et al., 2008, 2012). Interestingly, both EYFPPIP5K1 and PIP5K2-EYFP displayed pronounced polarized
distribution and localized predominantly to apical and basal
plasma membrane areas (Figure 5A). In some cases, this localization pattern was accompanied by nuclear localization (see
Supplemental Figure 7 online). To test whether polarized distribution observed for the overexpressed PI4P 5-kinases was
independently reflected by corresponding polarization of
7 of 18
PtdIns(4,5)P2, we analyzed the distribution of the lipid by monitoring YFP-PHPLCd1, a reporter that specifically binds PtdIns(4,5)
P2 (van Leeuwen et al., 2007). In plants not overexpressing
a PI4P 5-kinase, YFP-PHPLCd1 displayed polarized distribution to
apical and basal membrane domains in a pattern similar to that
found for EYFP-PIP5K1 and PIP5K2-EYFP (Figures 5A and 5B).
We quantified the polarization of EYFP-PIP5K1, PIP5K2-EYFP,
and of YFP-PH PLCd1 as ratios of lateral versus apicobasal
plasma membrane fluorescence, which were significantly less
than one in cells of both the root cortex and the root epidermis
(Figure 5C).
Figure 5. Polarized Distribution of PIP5K1, PIP5K2, and PtdIns(4,5)P2 in Root Cells.
EYFP-PIP5K1 or PIP5K2-EYFP was expressed in seedlings driven by an estradiol-inducible promoter and induction with 5 µM estradiol for 24 h. Images
were recorded by confocal microscopy. Images are representative of patterns observed in five independent transformation experiments. Bars = 10 µm.
AU, arbitrary units.
(A) Subcellular localization of overexpressed EYFP-PIP5K1 (left) and PIP5K2-EYFP (middle) in cells of root cortex (C) and epidermis (E), as indicated.
EYFP (right) is shown as a control. Top panels, fluorescence images; bottom panels, false-color representation indicating fluorescence intensities.
(B) Subcellular distribution of YFP-PHPLCd1, a fluorescent reporter for PtdIns(4,5)P2 in wild-type plants. Polarized distribution indicated by arrowheads.
Top panels, fluorescence images; bottom panels, false-color representation indicating fluorescence intensities.
(C) Ratio of lateral versus apicobasal plasma membrane fluorescence for EYFP-PIP5K1, PIP5K2-EYFP, and YFP-PHPLCd1 expressed in root cells, as
indicated. Plasma membrane–associated fluorescence of the membrane dye FM4-64 was used as a nonpolar control. C, cortex cells; E, epidermal
cells. Values <1 signify polarization toward the apicobasal sides. Data represent the mean ratios of 20 cells 6 SD. The asterisks indicate a significant
difference from the FM4-64 control according to a Student’s t test (*P < 0.05; **P < 0.01).
8 of 18
The Plant Cell
Plants used for localization studies were only mildly induced.
Consistent with overproduction of the PI4P 5-kinases, strongly
induced overexpressing lines accumulated increased amounts
of PtdIns(4,5)P2 (Figure 6A). When the EYFP-PIP5K1 or PIP5K2EYFP overexpressors were grown on vertical agar plates containing up to 5 µM estradiol, the plants exhibited a loss of
gravitropic growth concurrent with random nondirectional root
growth (Figures 6C and 6D). By contrast, estradiol treatment did
not affect the growth of control plants overexpressing EYFP
(Figure 6B). Random root growth was not observed in noninduced plants (data not shown). Auxin-related phenotypes of
the overexpressors were not limited to gravitropic bending.
Seven-day-old seedlings of all selected overexpressor lines
exhibited changes in root hair morphology after 24 h of incubation on agar plates containing 5 µM estradiol. Root hairs of
the induced overexpressors were shorter and thicker compared
with noninduced controls (Figures 5E, 5F, and 5H). In addition,
root hairs in the induced overexpressors often initiated at multiple sites of the trichoblasts (Figures 5G and 5I). The pattern
observed was similar to that previously reported for root hair–
specific overexpression of the PI4P 5-kinase isoform, PIP5K3
(Kusano et al., 2008; Stenzel et al., 2008) and resembles phenotypes described for plants with perturbed auxin distribution in
roots (Fischer et al., 2006). The effect on root hair morphology
likely results from the ectopic PI4P 5-kinase expression, as
PIP5K1 and PIP5K2 are normally expressed mainly in the vascular
Figure 6. Overexpression of PIP5K1 and PIP5K2 Results in Increased Levels of PtdIns(4,5)P2 and Auxin-Related Growth Defects.
(A) Accumulation of PtdIns(4,5)P2 in induced transgenics displaying strong induction, as indicated by fluorescence intensity detected for EYFP-PIP5K1
(left) and PIP5K2-EYFP (right). Data represent means 6 SD from three independent experiments. Asterisks represent significant differences in
PtdIns(4,5)P2 levels upon induced versus the noninduced controls according to a Student’s t test (*P < 0.05). AU, arbitrary units.
(B) to (D) Effects of overexpression of EYFP-PIP5K1 or PIP5K2-EYFP on root growth of 7-d-old seedlings. Patterns are representative for 24 plants for
each treatment. Seedlings were transferred to vertical plates containing 3 or 5 µM estradiol, as indicated. Plants were grown for 24 h and root growth
was documented. Arrows indicate the position of the root tip at the point of transfer. Bars = 1 mm.
(B) Control seedlings expressing EYFP.
(C) Seedlings expressing EYFP-PIP5K1.
(D) Seedlings expressing PIP5K2-EYFP.
(E) to (I) Arabidopsis plants expressing EYFP-PIP5K1 or PIP5K2-EYFP under an estradiol-inducible promoter were grown on solid MS medium for 7 d
and transferred onto media containing 5 µM estradiol for 24 h. Equally treated plants expressing EYFP were used as a control. Images were recorded by
bright-field or confocal microscopy. Images are representative for patterns observed in five independent transformation experiments. Bars = 200 µm in
(E), (F), and (H) and 50 µm in (G) and (I).
(E) Control expressing EYFP.
(F) and (G) EYFP-PIP5K1.
(H) and (I) PIP5K2-EYFP.
[See online article for color version of this figure.]
PtdIns(4,5)P2 Controls PIN Cycling
tissue of roots and only to a low degree in trichoblasts and root
hairs (Stenzel et al., 2008). No difference was observed between
the N-terminally tagged EYFP-PIP5K1 and the C-terminally tagged PIP5K2-EYFP with regard to subcellular localization, accumulation of PtdIns(4,5)P2, or any of the auxin-related phenotypes.
Polarization of PIN2-GFP Is Disturbed in the pip5k1 pip5k2
Double Mutant and upon Overexpression of PIP5K1
or PIP5K2
To test whether the phenotypes and changed auxin distribution
in the pip5k1 pip5k2 double mutant was linked to changed
patterns of PIN distribution, we crossed Arabidopsis lines expressing PIN1-GFP or PIN2-GFP into the pip5k1 pip5k2 double
mutant background and monitored the fluorescence distribution
of PIN1-GFP or PIN2-GFP in root tips (Figure 7). Clear polar
plasma membrane localization was observed in wild-type controls expressing PIN1-GFP (Figure 7A) or PIN2-GFP (Figure 7C).
The polarized patterns observed corresponded well with previously reported patterns and in cells of the central cylinder
PIN1-GFP localized predominantly to the basal side of the cells,
whereas PIN2-GFP localized predominantly to the apical sides
of cortex cells. In the pip5k1 pip5k2 double mutant background,
fluorescence intensities observed for PIN1-GFP and PIN2-GFP
were reduced. PIN1-GFP localized slightly more diffusely
compared with the sharply defined patterns found in wild-type
controls (Figure 7B). Plasma membrane association of PIN2GFP (Figure 7D) was not limited to the basal or apical sides of
the cells. Instead, in the double mutant, PIN2-GFP fluorescence
was also sometimes detected in lateral plasma membrane areas
(Figure 7D). These observations indicate perturbed polarization
of PIN proteins as a consequence of reduced levels of PtdIns
(4,5)P2. To test whether effects of overexpressing EYFP-PIP5K1
or PIP5K2-EYFP would also result in altered distribution of PIN
proteins, overexpressor lines were grown on vertical agar plates
and induced by the addition of estradiol. Plants displaying
agravitropic root growth (compared with Figures 6B to 6D) were
selected and the distribution of endogenous PIN1 and PIN2 was
analyzed using immunodetection by specific antibodies against
Arabidopsis PIN1 or PIN2 (Figures 7E to 7J). In estradiol-treated
plants expressing an EYFP control, PIN1 was detected at the
basal plasma membrane of cells of the central cylinder (Figure
7E), whereas PIN2 was detected at the apical plasma membrane
of cortex cells (Figure 7H), representing wild-type distribution.
PIN1 localization in the EYFP-PIP5K1 or PIP5K2-EYFP overexpressors resembled that found in the control plants (Figures
7F and 7G). PIN2 localization in the overexpressors differed from
that in the EYFP controls by consistently displaying weaker membrane association under identical conditions for immunodetection
and fluorescence imaging (Figures 7I and 7J). These observations
suggest that overexpression of EYFP-PIP5K1 or PIP5K2-EYFP
altered the membrane association of PIN2, which might result
from perturbed endocytic trafficking.
Endocytic Cycling of PIN1-GFP and PIN2-GFP Is Reduced in
the pip5k1 pip5k2 Double Mutant
To assess effects of reduced PtdIns(4,5)P2 levels on endocytic
cycling of PIN1-GFP and PIN2-GFP, we successively applied
9 of 18
cycloheximide (CHX) and brefeldin A (BFA), which inhibit de
novo protein synthesis and the reinsertion of membranous
compartments into the plasma membrane, respectively, resulting in the formation of BFA bodies. Care was taken not to exceed 25 µM BFA. When CHX and BFA were applied to wild-type
roots expressing PIN1-GFP (Figure 8A) or PIN2-GFP (Figure 8C),
BFA bodies decorated by PIN1-GFP or PIN2-GFP became visible within a few minutes and increased over 60 min, indicating
a substantial rate of endocytic recycling of PIN1-GFP and PIN2GFP between the plasma membrane and endosomal compartments. In sharp contrast, in roots of pip5k1 pip5k2 double mutants
expressing PIN1-GFP (Figure 8B) or PIN2-GFP (Figure 8D), we
observed fewer and smaller BFA bodies within 60 min of CHX/
BFA treatment. The data indicate that in the pip5k1 pip5k2
double mutant, endocytic cycling of PIN1-GFP and of PIN2-GFP
from the plasma membrane was impaired upstream of BFAsensitive endosomes. Overview images of CHX/BFA-treated
root tissue are provided in Supplemental Figure 8 online. To test
whether defects in endocytosis affected overall membrane internalization, wild-type controls, the pip5k1 and pip5k2 single
mutants, and the pip5k1 pip5k2 double mutant were subjected
to CHX/BFA treatment, stained with the styryl dye FM4-64, and
the subcellular distribution of the dye was monitored over a period of 90 min (Figures 8E and 8F; see Supplemental Figure 9
online). In all cases, the dye was internalized and decorated BFA
bodies. In the controls, the single mutants and the double
mutant the number of BFA bodies per cell increased with time
of dye exposure, indicating that membrane internalization was
essentially functional. Quantitative analysis (Figure 8G; see
Supplemental Figure 9B online) revealed a decreased number of
BFA bodies per cell in the mutant lines; for the pip5k1 pip5k2
double mutant, the reduction compared with wild-type controls
was significant after 50 and 90 min of dye application. The data
indicate that reduced levels of PtdIns(4,5)P2 impaired endocytosis of certain components, while other aspects remained
largely as in the wild type. This observation is in line with the
notion of alternative routes of endocytosis, only some of which
might depend on PtdIns(4,5)P2.
The Dynamics of Clathrin Light Chain Association with the
Plasma Membrane Are Altered in the pip5k1 pip5k2
Double Mutant
The endocytic cycling of PIN proteins from the plasma membrane
requires clathrin-mediated endocytosis (CME) (Dhonukshe et al.,
2007; Kitakura et al., 2011). Furthermore, CME has been linked
to phosphoinositides in tobacco (Nicotiana tabacum) pollen
tubes (Zhao et al., 2010). To further substantiate the hypothesis
that CME of PIN proteins requires PtdIns(4,5)P2 in Arabidopsis,
we monitored the dynamic membrane association of GFP-tagged
CLATHRIN LIGHT CHAIN 2 (CLC2-GFP) (Wang et al., 2013) in
7-d-old roots of wild-type controls and pip5k1 pip5k2 double
mutants by spinning disc confocal live imaging (Figure 9; see
Supplemental Movies 1 and 2 online). In wild-type controls,
CLC2-GFP decorated small punctae in the focal plane of the
plasma membrane and larger, fast-moving structures outside
the focal plane likely representing the trans-Golgi network (Figure 9A; see Supplemental Movies 1 and 2 online). The smaller,
10 of 18
The Plant Cell
plasma membrane–associated CLC2-GFP foci appeared in
wild-type controls at a mean density of 150 6 13 per 100 µm2
plasma membrane (Figure 9B) and were characterized by their
transient appearance and disappearance within a time interval
of ;26 6 2 s, as indicated by kymograph analysis (see
Supplemental Figure 10 online). In pip5k1 pip5k2 double mutants the plasma membrane–associated foci were present at
a substantially reduced density of only 63 6 12 per 100 µm2
plasma membrane (Figure 9C). Individual CLC2-GFP foci did not
display alterations in dwell time at the plasma membrane (;28 6
6 s; see Supplemental Figure 10 online) but appeared overall
much larger in the double mutant than in wild-type controls
(Figures 9B and 9D). The data indicate that recruitment of CLC2GFP to the plasma membrane of pip5k1 pip5k2 double mutants
was not impaired but that CLC2-GFP displayed altered distribution into fewer but larger plasma membrane–associated foci. This
observation is consistent with the notion that clathrin-dependent
internalization of PIN1-GFP and PIN2-GFP was impaired in the
pip5k1 pip5k2 double mutant (compared with Figure 8).
PIP5K1 and PIP5K2 Are Required for CCV-Associated
Formation of PtdIns(4,5)P2 and PI4P 5-Kinase Activity
Figure 7. Misexpression of EYFP-PIP5K1 or PIP5K2-EYFP Results in
Altered Localization and Polarization of PIN1 and PIN2.
(A) to (D) The subcellular distribution of PIN1-GFP and PIN2-GFP was
monitored by confocal fluorescence imaging in 10-d-old seedlings of
wild-type controls and the pip5k1 pip5k2 double mutant. Images are
representative for >10 independently grown plants. Bars = 10 µm.
(A) and (B) PIN1-GFP.
(A) Wild type control.
(B) pip5k1 pip5k2 double mutant.
(C) and (D) PIN2-GFP.
(C) Wild type control.
(D) pip5k1 pip5k2 double mutant.
(E) to (J) The localization of PIN1 and PIN2 was determined in induced
overexpressors of EYFP, EYFP-PIP5K1, or PIP5K2-EYFP by immunodetection
using specific antibodies against PIN1 (aPIN1) or PIN2 (aPIN2). Images are representative for >20 independently grown plants. Bars =
10 µm.
(E) and (H) EYFP control.
(F) and (I) EYFP-PIP5K1 overexpressor.
(G) and (J) PIP5K2-EYFP overexpressor.
(E) to (G) PIN1.
(H) to (J) PIN2.
To verify effects of PtdIns(4,5)P2 production on CLC2-GFP
dynamics (compared with Figure 9), we examined the rates
of PtdIns(4,5)P2 formation in subcellular fractions enriched for
CCVs. In Arabidopsis, salt stress can trigger increased formation
of CCVs (König et al., 2008b). Furthermore, the levels of PtdIns
(4,5)P2 in Arabidopsis increase upon salt treatment and associate with CCVs (König et al., 2008b). Therefore, we performed
salt stress experiments to test whether PIP5K1 and PIP5K2
contributed to the formation of CCV-associated PtdIns(4,5)P2
in Arabidopsis. Hydroponically grown wild-type controls and
pip5k1 and pip5k2 mutants were subjected to salt treatment by
the application of 0.4 M NaCl in culture medium, and CCVs were
isolated from leaves prior to stimulation and after 60 min, precisely as previously reported (König et al., 2008b). Only the
single pip5k1 and pip5k2 mutants could be used for hydroponic
growth, as the pip5k1 pip5k2 double mutants are too small to
provide the amount of material required for these tests. In wildtype plants, CCV-associated PI4P 5-kinase activity increased
upon salt stress (Figure 10), consistent with either translocation
of a PI4P 5-kinase from the plasma membrane to CCVs or with
activation of CCV-associated PI4P 5-kinase(s). By contrast, in
the pip5k1 mutant CCV-associated PI4P 5-kinase activity exhibited only a limited increase and no increase was detected in
the pip5k2 mutant upon stress treatment (Figure 10). The data
indicate that the CCV-associated PI4P 5-kinase activity is attributable largely to PIP5K2 and, to a lesser degree, PIP5K1. The
pattern is consistent with the in vitro catalytic activities of the
recombinant enzymes, as PIP5K2 displays five- to sixfold higher
activity than its close homolog, PIP5K1 (compared with
Supplemental Figure 6B online). The combined characterization
of CCVs formed upon salt treatment supports the notion that
Arrowheads, basal (PIN1) or apical (PIN2) localization. Arrows, mislocalization
or reduced membrane associated signal.
[See online article for color version of this figure.]
PtdIns(4,5)P2 Controls PIN Cycling
11 of 18
Figure 8. Endocytic Cycling of PIN1-GFP, PIN2-GFP, and FM 4-64 Is Reduced in the pip5k1 pip5k2 Double Mutant.
The dynamic distribution of PIN proteins was visualized in 10-d-old seedlings of wild-type controls and the pip5k1 pip5k2 double mutant expressing
PIN1-GFP or PIN2-GFP upon pretreatment for different times with 50 µM CHX and 25 µM BFA. The internalization of PIN1-GFP and PIN2-GFP into BFA
bodies was monitored in medial confocal sections of 2-µm thickness over a period of 60 min. Additionally, the internalization of the styryl dye FM4-64
was also monitored. Panels left to right, CHX/BFA treatment for 0, 30, and 60 min, as indicated. Bars = 10 µm.
(A) Wild-type controls, PIN1-GFP.
(B) pip5k1 pip5k2 double mutant, PIN1-GFP.
(C) Wild-type controls, PIN2-GFP.
(D) pip5k1 pip5k2 double mutant, PIN2-GFP.
(E) Wild-type controls, FM 4-64.
(F) pip5k1 pip5k2 double mutant, FM 4-64.
(G) The abundance of punctate signals per cell was quantified for all treatments based on digital imaging data for wild-type controls (black bars) and the
pip5k1 pip5k2 double mutant (white bars). Data represent mean 6 SD for 100 cells analyzed for each treatment. Asterisks indicate significant differences
from the wild type according to a Student’s t test (*P < 0.05; **P < 0.01).
[See online article for color version of this figure.]
12 of 18
The Plant Cell
mutants (Figure 2) indicate functional redundancy of PIP5K1 and
PIP5K2. This observation is consistent with the high sequence
similarity of PIP5K1 and PIP5K2 (Stenzel et al., 2012), which are
87% identical at the amino acid level, and with the similar expression patterns of PIP5K1 and PIP5K2 (see Supplemental
Figure 1 online). The mild phenotypes reported previously for
a pip5k2 single mutant have been attributed to altered auxin
distribution (Mei et al., 2012). However, the interpretation of data
reported for the single pip5k2 mutant (Mei et al., 2012) or obtained for both the pip5k1 and the pip5k2 mutants in this study is
complicated by the accompanying upregulation of at least one
other PI4P 5-kinase (Figure 1B), which might lead to compensatory effects or even overcompensation. Therefore, the characterization of the pip5k1 pip5k2 double mutant combined with
comprehensive lipid analysis is more informative and enables
interpretation of previously reported data (Mei et al., 2012). The
severe phenotype of the pip5k1 pip5k2 double mutant (Figures 2
and 3) indicates that PtdIns(4,5)P2 is a critical factor in plant
function and development. It must be noted that the severe
phenotype manifested in the pip5k1 pip5k2 double mutant despite a reduction in PtdIns(4,5)P2 levels by only ;40 to 50% in
these plants (Figures 1C and 1D). A possible explanation for this
effect arises from the notion of distinct functional pools of PtdIns
(4,5)P2 and the recent report that the regulatory consequences
of PtdIns(4,5)P2 in plant cells are partially directed by the identity
of the PI4P 5-kinase isoform generating the lipid (Stenzel et al.,
2012). Clearly, residual PtdIns(4,5)P2 generated in the pip5k1
pip5k2 double mutant by other PI4P 5-kinase isoforms was not
Figure 9. Altered Dynamics of Clathrin Light Chain Association with the
Plasma Membrane in the pip5k1 pip5k2 Double Mutant.
The dynamic association of clathrin with the plasma membrane was
monitored by spinning disc confocal live imaging in 7-d-old roots of wildtype controls and pip5k1 pip5k2 double mutants expressing CLC2-GFP.
Images presented are stills from Supplemental Movies 1 and 2 online.
(A) Plasma membrane foci decorated by CLC2-GFP in wild-type controls
(left) and the pip5k1 pip5k2 double mutant (right). Bars = 3 µm.
(B) Individual foci were analyzed for size. Representative foci shown are
marked in (A) by arrowheads.
(C) The density of CLC2-EYFP foci per plasma membrane area was calculated for wild-type controls and pip5k1 pip5k2 double mutants. Data
represent the mean 6 SD of six independent experiments. Asterisks indicate
a significant difference according to a two-tailed Student’s t test (**P < 0.01).
(D) Foci size was quantified for wild-type controls and pip5k1 pip5k2
double mutants. Data represent the mean 6 SD of six independent experiments. Asterisks indicate a significant difference according to a twotailed Student’s t test (**P < 0.01).
PIP5K1 and PIP5K2 associate with CCVs, consistent with a role
for PtdIns(4,5)P2 in CME.
DISCUSSION
The strong phenotype of the pip5k1 pip5k2 double mutant and
less pronounced phenotypes of the individual pip5k1 or pip5k2
Figure 10. PIP5K1 and PIP5K2 Are Required for Salt-Induced CCVAssociated Increases in PI4P 5-Kinase Activity.
Arabidopsis wild-type controls and the pip5k1 and pip5k2 mutants were
grown in hydroponic culture and subjected to salt treatment by 0.4 M
NaCl in liquid culture medium. CCVs were isolated before and after 60 min
of treatment, and the associated PI4P 5-kinase activity was assayed in
the CCV fractions using exogenous phosphatidylinositol 4-phosphate as
a substrate. Because the pip5k1 pip5k2 double mutant only yields limited
amounts of material and due to the large amount of material required for
CCV preparation, this experiment was performed only with single mutants. Data represent means 6 SD from three independent experiments.
Asterisks represent significant differences in the activity after stimulation
versus the nonstimulated controls according to a Student’s t test (*P <
0.05; **P < 0.01).
PtdIns(4,5)P2 Controls PIN Cycling
able to compensate for the lack of PIP5K1 and PIP5K2, possibly
because it represents one or more PtdIns(4,5)P2 pools that may
not be involved in controlling CME.
In line with previous observations on the pip5k2 single mutant
(Mei et al., 2012), several phenotypic aspects of the pip5k1
pip5k2 double mutant are consistent with an effect of PtdIns(4,5)P2
on auxin signaling, including impaired root growth (Figures 2B
and 2C), reduced apical dominance (Figure 2D), and the attenuated response to gravistimulation (Figure 3). In our experiments, auxin levels in whole double mutant seedlings were not
different from those in wild-type controls (Figure 4A). Auxin
measurements, particularly on the pip5k1 pip5k2 double mutant,
carried a large experimental error. Considering the experimental
difficulty in measuring auxin levels, we find our observation
consistent with data by Mei et al. (2012), who presented levels of
;8 ng g21 fresh weight in a pip5k2 single mutant as differing
from ;10 ng g21 fresh weight in wild-type controls. Based on
the auxin transport assays and two different DR5-based reporters (Figure 4), our data suggest that auxin transport and
distribution, rather than auxin amount, were perturbed in the
pip5k1 pip5k2 double mutant.
PIN1 is largely responsible for the basipetal auxin transport in
the central cylinder, whereas PIN2 mediates the upward transport of auxin in the cortex and epidermis of the root tip and the
asymmetric distribution of auxin during gravitropic responses
(Sukumar et al., 2009; Rahman et al., 2010). Together, PIN1 and
PIN2 contribute to the establishment of the basipetal auxin
gradient in the root tip (Grieneisen et al., 2007). Therefore, we
focused on the effects of PtdIns(4,5)P2 on the distribution of
PIN1-GFP and PIN2-GFP. The influence of PtdIns(4,5)P2 on the
distribution of PIN2-GFP is of particular interest in light of the
previously reported involvement of phosphoinositides in gravitropic signaling in different plant species (Perera et al., 1999,
2001, 2006). The perturbation of PIN1-GFP distribution in the
pip5k1 pip5k2 double mutant (Figure 7B) was not limited to
a polarization defect, but rather the protein was not efficiently
inserted into the plasma membrane, suggesting a defect in
secretory membrane trafficking. This observation is consistent
with the report that Arabidopsis PIP5K2 interacts with Rab-E
isoforms implicated in the control of Golgi–to–plasma membrane
trafficking (Camacho et al., 2009) and suggests a function for
PIP5K1 and PIP5K2 in Rab-E–dependent secretion of PIN1-GFP.
Considering how PtdIns(4,5)P2 might contribute to the control
of auxin transport, it must also be noted that PtdIns(4,5)P2 has
been demonstrated to mediate clathrin recruitment in plant cells
(König et al., 2008b; Zhao et al., 2010) and that polarization and
PIN cycling involves clathrin-mediated membrane trafficking
(Dhonukshe et al., 2007; Kitakura et al., 2011). The link to CME
suggests that reduced PtdIns(4,5)P2 levels would influence both
clathrin dynamics and PIN recycling, and this notion is supported by this study (Figures 8 to 10). While expression of
fluorescence-tagged variants of PIP5K1 and PIP5K2 under their
respective intrinsic promoters resulted in complementation of
the mutant phenotype (compared with Figure 2), no fluorescence was detected in the complemented plants, likely due to
low promoter activity or to limited stability of the PI4P 5-kinase
proteins in planta. The polarized subcellular localization of
overexpressed EYFP-PIP5K1 and PIP5K2-EYFP in cells of the
13 of 18
root tip (Figures 5A and 2C) is consistent with an effect on PIN
cycling. While it cannot be ruled out that the localization patterns
observed were biased by the overexpression, it must be noted
that the YFP-PHPLCd1 reporter for PtdIns(4,5)P2 displayed similar
polarized distribution in plants not overexpressing a PI4P 5-kinase
(Figures 5B and 5C). Together, the data indicate that PIP5K1
and PIP5K2 generate PtdIns(4,5)P2 in polarized apical and basal
plasma membrane microdomains important for the asymmetric
distribution of PIN proteins. The failure to form BFA bodies
decorated by PIN1 or PIN2 and the observation of fewer but
larger plasma membrane CLC2-GFP foci in the pip5k1 pip5k2
double mutant (Figure 9) suggest that it is not clathrin recruitment to the membrane but rather cleavage of CCVs from
the membrane that might be perturbed in these plants. In the
double mutants with reduced PtdIns(4,5)P2 levels clathrin patches
might appear larger because fewer CCVs are released and
membrane-associated CLC2-GFP foci might coalesce. This concept is consistent with our understanding how CCV formation is
mediated by dynamins in a PtdIns(4,5)P2-dependent fashion in
mammalian cells (Lemmon and Ferguson, 2000).
The involvement of PtdIns(4,5)P2 in the control of plasma
membrane cycling of PIN proteins ties in with reports on other
membrane constituents. For instance, effects on PIN cycling
have been independently attributed also to sterols (Men et al.,
2008) and sphingolipids containing very-long-chain fatty acids
(Markham et al., 2011), leading us to speculate that PtdIns(4,5)P2,
sterols, and sphingolipids might synergistically define membrane
domains required for PIN recycling. Association of PtdIns(4,5)P2
with sphingolipids in detergent-insoluble membrane preparations has previously been described (Furt et al., 2010), but no
biological function had been attributed to the association. As
dynamins must exert a physical force to cleave off a CCV from
the plasma membrane, this force requires a counterforce in the
membrane bilayer. It appears possible that sterols, sphingolipids, and PtdIns(4,5)P2 form a joint membrane microdomain with
particular stabilizing properties that might be required for
dynamin function, as has been proposed for membrane microdomains involved in perceiving mechanical force (Anishkin and
Kung, 2013).
Besides controlling CME, PtdIns(4,5)P2-containing membrane
domains might also influence PIN recycling through indirect effects on PIN phosphorylation. For instance, PtdIns(4,5)P2 might
act as a membrane-lipid ligand to activate a protein phosphorylation cascade consisting of 3-phosphoinositide-dependent protein kinase 1, which contains a Pleckstrin homology (PH) domain
and phosphorylates the protein kinase PINOID (PID), thereby
activating it (Zegzouti et al., 2006). Activated PID in turn phosphorylates PIN proteins, and this phosphorylation is required for
their dynamic polar distribution (Robert and Offringa, 2008). In this
context, enlarged CLC2-GFP foci in the plasma membrane might
also arise from incorrect recruitment of CCVs into apically or
basally bound sorting pathways. The data presented here are
thus also consistent with a mechanism where PtdIns(4,5)P2
controls PIN-localization through effects on the PIN phosphorylation machinery, as was previously proposed (Zegzouti et al.,
2006), in analogy to the polar localization of Glc transporters
mediated by protein kinases B and C in mammalian cells (Watson
et al., 2004).
14 of 18
The Plant Cell
The dwarf phenotype of the pip5k1 pip5k2 double mutant
suggests that one consequence of reduced PtdIns(4,5)P2 formation is a defect in the balance of cell proliferation and cell
differentiation. Because auxin promotes stem cell proliferation
(Dello Ioio et al., 2008) and a dwarf phenotype implies a shift
toward differentiation, the dwarf phenotype observed in the
pip5k1 pip5k2 double mutant is consistent with impaired distribution of auxin. Thus, altered growth in consequence of the
reduced PtdIns(4,5)P2 production is consistent with a role of
PtdIns(4,5)P2 in the control of directional auxin transport in root
tips (Bennett and Scheres, 2010). While effects of PtdIns(4,5)P2
on the plasma membrane association of clathrin and, thus, the
polar distribution of PIN proteins were evident, other facets of
the pip5k1 pip5k2 double mutant phenotype may have additional causes not related to auxin. The pip5k1 pip5k2 double
mutant reported may thus represent a tool to further dissect
regulatory roles of PtdIns(4,5)P2 in Arabidopsis.
METHODS
cDNA Constructs
Constructs for Stable Expression in Arabidopsis
Amplification of 1500-bp genomic sequences upstream of coding
sequences for use as promoters was achieved with different Arabidopsis
thaliana bacterial artificial chromosome clone templates as indicated,
using the primer combinations PromPIP5K1for/PromPIP5K1rev on BAC
clone F2E2 and PromPIP5K2for/PromPIP5K2rev on bacterial artificial
chromosome clone T32E8. The PCR products representing promoter
regions of PIP5K1 or PIP5K2 were moved directionally as SalI-NotI fragments into the vector pUC18Entry-EYFP, creating the plasmids PromPIP5K1-EYFP and PromPIP5K2-EYFP. The coding regions of PIP5K1
and PIP5K2 were amplified from PIP5K1- pGem-T-easy or PIP5K2pGem-T-easy (Stenzel et al., 2008), respectively, using the primer
combinations PIP5K1for/PIP5K1rev and PIP5K2for/PIP5K2rev. The
coding sequences for PIP5K1 and PIP5K2 were moved as NotI-NotI
fragments from PIP5K1-pGEM-Teasy and PIP5K2-pGEM-Teasy into
PromPIP5K1-EYFP and PromPIP5K2-EYFP, respectively. A stop codon
between PIP5K1/ PIP5K2 and the respective EYFP reading frames was
removed by mutagenesis using the primers QCPIPK1for/QCPIPK1rev
and QCPIPK2for/QCPIPK2rev. Finally, the fragments consisting of the
respective promoter sequence and the fusion constructs encoding
PIP5K1-EYFP and PIP5K2-EYFP, respectively, were transferred to the
expression plasmid pCAMBIA3300.0 GC using Gateway technology
(Invitrogen) according to the manufacturer’s instructions.
Cloning of PI4P 5-Kinase Promoter Sequences for Generation
of Promoter-GUS Fusions
The same PCR products representing PromPIP5K1 and PromPIP5K2 as
used above were moved directionally as SalI-NotI fragments into the
vector pgreenGUSPlus (Stenzel et al., 2008). The resulting plasmids were
transformed into Agrobacterium tumefaciens strain EHA105 and used for
stable Arabidopsis transformation.
(Promega), it was moved as a NotI-NotI fragment into the vector YEp112A1NEX (provided by Ellen Hornung, Georg-August-University, Goettingen).
Fusion Constructs for Inducible Overexpression in Arabidopsis
The coding region for PIP5K1 was moved from PIP5K1-pGem-T-easy into
the plasmid pUC18Entr-ccdb as a NotI-NotI fragment. This introduced
a new NcoI site contained in the primer sequence that was used for the
introduction of the EYFP coding region, previously amplified using the
primer combination EYFPfor/EYFPrev.
The coding region for PIP5K2 was moved from PIP5K2-pGem-T-easy
as a SalI-NotI fragment into pUC18Entry-EYFP. Both EYFP-PIP5K1 and
PIP5K2-EYFP were then transferred to the expression plasmid pMDC7
(Curtis and Grossniklaus, 2003) using Gateway technology (Invitrogen)
according to the manufacturer’s instructions.
All primer sequences are specified in Supplemental Table 1 online.
Arabidopsis Culture
Experiments were performed with Arabidopsis ecotype Columbia-0 (Col-0)
or in Col-0 plants carrying T-DNA insertions in pipk5k1 (SALK_146728) and/or
for pip5k2 (SALK_012487). For stress treatments, seeds were surface
sterilized by incubation for 15 min at 22°C in 6% (w/v) sodium hypochlorite
in 0.1% Triton X-100 and washed five times in sterile distilled water. Seeds
were vernalized at 4°C overnight and cultured in sealed jars on 0.5%
Murashige and Skoog (MS) medium with modified vitamins (Duchefa)
containing 1% Suc and 0.25% Gelrite (Carl Roth). After 14 d, plants were
transferred into hydroponic cultures in liquid medium according to Randall
and Bouma (1973). Hydroponic cultures were exposed to 140 mmol
photons m22 s21 of light under a light/dark regime of 8 h light/16 h dark and
continuous aeration. For stress treatments, 8- to 10-week-old-plants were
stimulated by adding NaCl to a final concentration of 0.4 M to the hydroponic medium. Rosette leaves were harvested before treatment and
after various periods of stimulation and immediately frozen in liquid nitrogen.
Arabidopsis Transformation
Recombinant constructs were introduced into Arabidopsis lines using the
floral dip method (Clough and Bent, 1998). Independent transformants
were selected on MS media containing 50 µg mL21 kanamycin or 15 µg mL21
hygromycin, 1% (w/v) Suc, and 0.6% (w/v) agar or grown on soil and sprayed
after 7 d with a 0.5% (w/v) Basta solution (Bayer Crop Science).
CCV Isolation
CCVs were enriched according to Harley and Beevers (1989) with minor
modifications as previously described (König et al., 2008b). To prevent
protein degradation, Pefabloc (Carl Roth) and protease inhibitor cocktail
(Carl Roth) were included in all buffers.
Extraction and Quantification of Lipids
Lipids were extracted and analyzed according to König et al. (2008a) with
slight modifications, as fatty acid methyl esters were analyzed using a
30 m 3 250-µm DB-23 capillary column (Agilent) and a GCMS-QP2010S-EI
gas chromatography–mass spectrometry system (Shimadzu).
Radiodetection of PtdIns(4,5)P2
Constructs for Stable Expression in Saccharomyces cerevisiae
PIP5K1 and PIP5K2 were moved as NotI-NotI fragments from PIP5K1- or
PIP5K2-pGem-T-easy, respectively, into the yeast expression vector. Mss4
was amplified from S. cerevisiae cDNA using the primer combination
MSS4for/MSS4rev. After cloning the PCR product into pGEM-T-easy
Twelve-day-old Arabidopsis seedlings grown on MS agar plates were
transferred to a 24-well plate and equilibrated overnight under continuous
light with gentle shaking. Each well contained 500 mL of MS media with
1% Suc and six Arabidopsis seedlings (for wild-type and single mutant
lines) and eight seedlings (pipk1 pipk2 double mutant). Radiolabel (50 mCi
PtdIns(4,5)P2 Controls PIN Cycling
of [32P]Pi) was added to each well, and seedlings were harvested after
30 min, blotted on Kimwipes, and incubated for 20 min on ice in 500 mL of
cold 20% perchloric acid. Seedlings were transferred to glass tubes, and
lipids were extracted and separated by thin layer chromatography as
previously described (Perera et al., 2005). The amount of [32P]Pi-labeled
PtdInsP2 was calculated as the percentage of the total [32P]Pi incorporated into the lipid phase of each sample.
Lipid Kinase Assays
Lipid kinase activity was determined as previously described (Cho and
Boss, 1995). Reaction products were identified according to comigration
with authentic standards (Avanti Polar Lipids) and quantified on thin layer
chromatography plates by phosphor imaging (Phosphoimager FLA-3000;
Fujifilm).
Heterologous Expression in S. cerevisiae
PI4P 5-kinases were expressed in S. cerevisiae strain YOC808 and
complementation tests performed as previously described (Homma et al.,
1998). The parental strain, YOC807, was used as a control.
Determination of Gravitropic Bending
Gravitropic bending was determined using plants grown on MS media
in vertical Petri dishes as previously described (Fukaki et al., 1996)
according to digital images.
Genotyping
The absence or presence of T-DNA inserts was detected by PCR on
genomic DNA templates. The following primer combinations were used
to amplify the wild-type alleles: gPIPK1for/gPIPK1rev and gPIPK2for/
gPIPK2rev. Mutant alleles were amplified using LBa1 and the respective
reverse primer.
Real-Time RT-PCR Analysis
Total RNA was extracted using plant RNA purification reagent (Invitrogen).
RNA was incubated with RNase-free DNase I for 30 min at 37°C to remove
genomic DNA contamination, and RNA was ethanol precipitated. Five
micrograms of total RNA was used as a template for reverse transcription
with RevertAid H Minus M-MuLV reverse transcriptase (Fermentas) in the
presence of oligo(dT) primers. Equal amounts of first-strand cDNAs were
used as templates for real-time PCR amplification using the following
primer combinations: qPIP5K1for/qPIP5K1rev and qPIP5K2for/qPIP5K2rev.
The Arabidopsis actin gene, ACT2, was amplified using primer combination
ACT2for/ACT2rev. Quantitative real-time PCRs were performed in a MiniOpticon System (Bio-Rad), and PIP5K1 and PIP5K2 transcript levels were
quantified in relation to ACT2 levels.
Determination of Auxin Levels
Plant material was extracted as previously described (Matyash et al.,
2008). The analysis of constituents was performed using an Agilent 1100
HPLC system (Agilent Technologies) coupled to an Applied Biosystems
3200 hybrid triple quadrupole/linear ion trap mass spectrometer (MDS
Sciex). Nanoelectrospray ionization (nanoESI) analysis was achieved
using a chip ion source (TriVersaNanoMate; AdvionBioSciences). Reversed-phase HPLC separation was performed on an EC 50/2 Nucleodure C18 gravity 1.8-µm column (50 3 2.1 mm, 1.8-µm particle size;
Macherey and Nagel), applying a column temperature of 30°C. For
analysis, 10 mL of extract was injected. The binary gradient system
consisted of solvent A, water/acetic acid (100:0.1, v/v), and solvent B,
acetonitrile/acetic acid (100:0.1, v/v), with the following gradient program:
15 of 18
5% solvent B for 1 min, followed by a linear increase of solvent B up to
95% within 10 min and an isocratic run at 95% solvent B for 4 min. To
reestablish starting conditions, a linear decrease to 5% B within 2 min was
performed, followed by 10-min isocratic equilibration at 5% B. The flow
rate was 0.3 mL min21. For stable nanoESI, 130 mL min21 of 2-propanol/
acetonitrile/water/acetic acid (70:20:10:0.1, v/v/v/v) delivered by a 2150
HPLC pump (GE Healthcare) was added just after the column via a mixing
tee valve. Using another post column splitter, 790 nL min21 of the eluent
was directed to the nanoESI chip. Ionization voltage was set to 21.7 kV.
IAA was negatively ionized and determined in a scheduled multiple reaction monitoring mode. For the scheduled mode, the multiple reaction
monitoring detection window was set to 72 s and a target scan time of
1,2 s was applied. Mass transitions were as follows: for D5-IAA 179/135
(declustering potential 240 V, entrance potential 26.5 V, and collision
energy 222 V) and for IAA 174/130 (declustering potential 255 V, entrance potential 24 V, and collision energy 216 V). The mass analyzers
were adjusted to a resolution of 0.7 atomic mass units full width at half
height. The ion source temperature was 40°C, and the curtain gas was set
at 10 (given in arbitrary units). Quantification was performed using a calibration curve of intensity (mass-to-charge) ratios of [unlabeled]/[deuteriumlabeled] versus molar amounts of unlabeled (0.3 to 1000 pmol).
Determination of Auxin Transport
Seven-day-old Arabidopsis Col-0 and 10-d-old day old pip5k1 pip5k2
seedlings with similar root lengths were grown on vertical plates. A piece
of agarose gel containing 10 µM IAA (Sigma-Aldrich) and 100 nM [3H]IAA
(Radiochemicals) was placed at the juncture between root tip and hypocotyl. Auxin was allowed to diffuse into the root. After 18 h, the terminal
1-cm tip of the root was removed and radioactivity determined by liquid
scintillation counting using an Analyzer Tricarb 1900 TR (Canberra
Packard).
Staining, Fixing, or Application of Pharmacological or
Immunological Agents
CHX and BFA Treatments
Plants were treated with 50 µM CHX ;15 min prior to imaging. For the
PIN1-GFP and PIN2-GFP recycling kinetic analysis, 25 µM BFA was
applied to the plantlets, and confocal images were taken after the times
indicated.
Whole-Mount Immunolabeling of PIN Proteins
Whole-mount immunolabeling of root tips was done as previously described (Sauer et al., 2006) with modifications: After fixation, root tips were
mounted on poly-L-Lys–coated slides, air-dried, and stored at 220°C.
After rehydration and cell wall digestion, blocking was done with 5% BSA
in PBS, and antibodies were diluted in 5% BSA/PBS supplemented with
1% acetylated BSA (Aurion). Goat anti-PIN1 (ap-20, diluted 1:250; Santa
Cruz Biotechnology) and chicken anti-PIN2 (diluted 1:250; Agrisera) were
used as primary antibodies. Donkey anti-goat IgG coupled to AlexaFluor488
and goat anti-chicken IgG coupled to AlexaFluor488 (all from Invitrogen)
diluted 1:500 were used as secondary antibodies.
FM4-64 (Molecular Imaging Products) was added to root tips at a final
concentration of 10 µM as described (Parton et al., 2001) and visualized
after different periods of incubation.
Histochemical Staining for GUS Activity
Histochemical staining of plant tissue for GUS activity was performed as
previously described (Jefferson et al., 1987). GUS-positive samples were
16 of 18
The Plant Cell
examined with a bright-field microscope (Olympus BX51) or a stereomicroscope (Olympus SZX12) at low magnification (34 to 310), both
equipped with a camera, and digital images were recorded.
Supplemental Figure 4. Elongation of Hypocotyls and Roots for
Gravitropism Tests in pip5k1 and pip5k2 Single Mutants and the
pip5k1 pip5k2 Double Mutant.
Microscopy and Imaging
Supplemental Figure 5. Auxin Distribution Is Impaired in the pip5k1
pip5k2 Double Mutant.
For root length and leaf area measurements, plants were imaged using
a flatbed scanner (Canonscan 800S; Canon). Lengths of roots and
hypocotyls were determined using ImageJ (v. 1.41; National Institutes of
Health), while leaf area was calculated using BlattFlaeche software
(DatInf). Bright-field and epifluorescence images were recorded using an
Olympus BX51 microscope, a F41-028 HQ filter set for YFP (Olympus),
a F41-007 HQ filter set for Redstar, an Olympus ColorView II camera, and
analySISDocu3.2 software (Soft-Imaging-Systems). Alternatively, images
were recorded using a Zeiss AxioImager M1 microscope with filter sets 43
for DsRed, 46 for EYFP, and 47 for CFP. Confocal images were recorded
using a Zeiss LSM 510 confocal microscope. EYFP was excited at 514 nm
and imaged using an HFT 405/514/633-nm major beam splitter and
a 529- to 603-nm band-pass filter. EYFP and Redstar were simultaneously excited at 488 and 561 nm, respectively, and imaged using a 405/
488/561-nm major beam splitter and 505- to 550-nm and 575- to 615-nm
band-pass filters. Alternatively, confocal microscopy was performed
using an LSM 710 confocal microscope (Zeiss). Multi Time macro (Multi
Time Series PLUS ZEN 2010-18) was used for automated series of multiple
roots. Images were processed and analyzed using Fiji software (http://pacific.
mpi-cbg.de/). Three-dimensional images of whole root tips were reconstructed
using the Fiji Stitching plug-in (Preibisch et al., 2009). Immunolabeled root tips
were analyzed using an LSM700 (Zeiss). Immunomicrographs were processed
through Photoshop 12.0.4 program (Adobe).
The fluorescently tagged CLC2-GFP marker lines were imaged on
a confocal microscope equipped with a Yokogawa spinning disc head
fitted to a Nikon Ti-E inverted microscope, a total internal reflected
fluorescence (TIRF) oil immersion objective (Nikon CFI Apo TIRF 3 100,
numerical aperture of 1.49) an evolve charge-coupled device camera
(Photometrics Technology), and a 31.2 lens between the spinning disc
and the camera. GFP was excited at 491 nm using a multichannel dichroic
and an ET525/50M band-pass emission filter (Chroma Technology), while
exposure time was 800 ms for the GFP laser. Image acquisitions were
performed using Metamorph online premier (version 7.5). Images were
processed with the help of Fiji image analysis software. Background
correction was performed using subtract background (rolling ball radius
20 pixels), and walking average plug-ins in Fiji were used to smoothen the
movies. Kymographs were obtained using the multiple kymograph plugin, while clathrin density measurements were performed using the Fiji
plug-in TrackMate.
Supplemental Figure 6. Fluorescence-Tagged PIP5K1 and PIP5K2
Act as PI4P 5-Kinases and Complement a Yeast MSS4 Mutant.
Supplemental Figure 7. Fluorescence-Tagged Variants of PIP5K1
and PIP5K2 Localize to the Plasma Membrane and the Nucleus of
Arabidopsis Cells.
Supplemental Figure 8. Endocytic Cycling of PIN1-GFP and PIN2GFP Is Reduced in the pip5k1 pip5k2 Double Mutant.
Supplemental Figure 9. Endocytic Cycling of the Styryl Dye FM 4-64
Is Slightly Reduced in the pip5k1 and pip5k2 Single Mutants.
Supplemental Figure 10. Lifetime Analysis of Plasma Membrane–
Associated Clathrin Foci.
Supplemental Table 1. Primer Sequences.
Supplemental Movie 1. Dynamics of CLC2-GFP in Root Cells of
Arabidopsis Wild Type.
Supplemental Movie 2. Dynamics of CLC2-GFP in Root Cells of the
Arabidopsis pip5k1 pip5k2 Double Mutant.
ACKNOWLEDGMENTS
We thank Jiri Friml (Institute of Science and Technology Austria) for
Arabidopsis lines expressing fluorescently labeled PIN proteins and for
helpful discussion. We also thank Sebastian Bednarek (University of
Wisconsin–Madison) for providing Arabidopsis lines expressing fluorescently
labeled CLC, Teun Munnik (University of Amsterdam, The Netherlands) for
the YFP-PHPLCd1 reporter plants, and Yoshikazu Ohya (University of
Tokyo, Japan) for the mss4TS yeast strains. We thank Ellen Hornung
(Georg-August-University Goettingen, Germany) for plasmids. This work
was supported by Grants He3424/1 and He3424/2 from the German
Research Foundation (Deutsche Forschungsgemeinschaft; to I.H.), by
Grant MCB0718452 from the National Science Foundation (to W.F.B.
and I.Y.P.), and by a European Molecular Biology long-term fellowship
(EMBO-ALTF 299-2010, to T.I.). S.P. was supported by the Max-Planck
Society, and P.K. was supported by the European Union Seventh Framework
Programme (FP7 2007-2013) under Grant Agreement 263916 (WallTrac,
Marie Curie Initial Training Network).
Accession Numbers
AUTHOR CONTRIBUTIONS
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: ACT2, At3g18780; CLC2, At2g40060; PIP5K1, At1g21980;
PIP5K2, At1g77740; PIN1, At1g73590; and PIN2, At5g57090.
I.H. designed research, analyzed data, and wrote the article. T.Isch.,
S.W., P.K., and J.L. designed research, performed experiments, analyzed data, and edited the article. M.M., I.S., C.L., T.W., and T.Iv.
performed experiments and analyzed data. Y.J.I., I.Y.P., and B.H.
performed experiments, analyzed data, and edited the article. W.B., T.T.,
W.F.B., I.F., and S.P. analyzed data and edited the article.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. PIP5K1 and PIP5K2 Are Expressed in Various
Organs of Arabidopsis.
Supplemental Figure 2. T-DNA Insertion Mutants Deficient in PIP5K1
and PIP5K2 Have Unaltered Levels of Structural Phospholipids.
Supplemental Figure 3. T-DNA Insertion Mutants with the Genotypes
pip5k1(2/2)pip5k2(+/2) or pip5k1(+/2)pip5k2(2/2) Are Phenotypic Intermediates between Single and Double Mutants.
Received July 24, 2013; revised September 22, 2013; accepted October
15, 2013; published December 10, 2013.
REFERENCES
Anishkin, A., and Kung, C. (2013). Stiffened lipid platforms at
molecular force foci. Proc. Natl. Acad. Sci. USA 110: 4886–4892.
PtdIns(4,5)P2 Controls PIN Cycling
Balla, T. (2006). Phosphoinositide-derived messengers in endocrine
signaling. J. Endocrinol. 188: 135–153.
Bennett, T., and Scheres, B. (2010). Root development-two
meristems for the price of one? Curr. Top. Dev. Biol. 91: 67–102.
Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J.,
Heidstra, R., Aida, M., Palme, K., and Scheres, B. (2005). The PIN
auxin efflux facilitator network controls growth and patterning in
Arabidopsis roots. Nature 433: 39–44.
Boutté, Y., Crosnier, M.T., Carraro, N., Traas, J., and Satiat-Jeunemaitre,
B. (2006). The plasma membrane recycling pathway and cell polarity in
plants: Studies on PIN proteins. J. Cell Sci. 119: 1255–1265.
Camacho, L., Smertenko, A.P., Pérez-Gómez, J., Hussey, P.J., and
Moore, I. (2009). Arabidopsis Rab-E GTPases exhibit a novel
interaction with a plasma-membrane phosphatidylinositol-4-phosphate
5-kinase. J. Cell Sci. 122: 4383–4392.
Cho, M.H., and Boss, W.F. (1995). Transmembrane signaling and
phosphoinositides. Methods Cell Biol. 49: 543–554.
Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana.
Plant J. 16: 735–743.
Curtis, M.D., and Grossniklaus, U. (2003). A Gateway cloning vector
set for high-throughput functional analysis of genes in planta. Plant
Physiol. 133: 462–469.
Dello Ioio, R., Nakamura, K., Moubayidin, L., Perilli, S., Taniguchi,
M., Morita, M.T., Aoyama, T., Costantino, P., and Sabatini, S.
(2008). A genetic framework for the control of cell division and
differentiation in the root meristem. Science 322: 1380–1384.
Dhonukshe, P., Aniento, F., Hwang, I., Robinson, D.G., Mravec, J.,
Stierhof, Y.D., and Friml, J. (2007). Clathrin-mediated constitutive
endocytosis of PIN auxin efflux carriers in Arabidopsis. Curr. Biol.
17: 520–527.
Dhonukshe, P., et al. (2008). Generation of cell polarity in plants links
endocytosis, auxin distribution and cell fate decisions. Nature 456:
962–966.
Elge, S., Brearley, C., Xia, H.J., Kehr, J., Xue, H.W., and MuellerRoeber, B. (2001). An Arabidopsis inositol phospholipid kinase
strongly expressed in procambial cells: Synthesis of PtdIns(4,5)P2
and PtdIns(3,4,5)P3 in insect cells by 5-phosphorylation of precursors.
Plant J. 26: 561–571.
Fischer, U., Ikeda, Y., Ljung, K., Serralbo, O., Singh, M., Heidstra,
R., Palme, K., Scheres, B., and Grebe, M. (2006). Vectorial
information for Arabidopsis planar polarity is mediated by combined
AUX1, EIN2, and GNOM activity. Curr. Biol. 16: 2143–2149.
Fukaki, H., Fujisawa, H., and Tasaka, M. (1996). Gravitropic
response of inflorescence stems in Arabidopsis thaliana. Plant
Physiol. 110: 933–943.
Furt, F., König, S., Bessoule, J.J., Sargueil, F., Zallot, R., Stanislas,
T., Noirot, E., Lherminier, J., Simon-Plas, F., Heilmann, I., and
Mongrand, S. (2010). Polyphosphoinositides are enriched in plant
membrane rafts and form microdomains in the plasma membrane.
Plant Physiol. 152: 2173–2187.
Grieneisen, V.A., Xu, J., Marée, A.F., Hogeweg, P., and Scheres, B.
(2007). Auxin transport is sufficient to generate a maximum and
gradient guiding root growth. Nature 449: 1008–1013.
Harley, S.M., and Beevers, L. (1989). Coated vesicles are involved in
the transport of storage proteins during seed development in Pisum
sativum L. Plant Physiol. 91: 674–678.
Heilmann, I. (2009). Using genetic tools to understand plant
phosphoinositide signalling. Trends Plant Sci. 14: 171–179.
Homma, K., Terui, S., Minemura, M., Qadota, H., Anraku, Y.,
Kanaho, Y., and Ohya, Y. (1998). Phosphatidylinositol-4phosphate 5-kinase localized on the plasma membrane is essential
for yeast cell morphogenesis. J. Biol. Chem. 273: 15779–15786.
17 of 18
Ischebeck, T., Seiler, S., and Heilmann, I. (2010a). At the poles
across kingdoms: Phosphoinositides and polar tip growth.
Protoplasma 240: 13–31.
Ischebeck, T., Stenzel, I., and Heilmann, I. (2008). Type B
phosphatidylinositol-4-phosphate 5-kinases mediate Arabidopsis
and Nicotiana tabacum pollen tube growth by regulating apical
pectin secretion. Plant Cell 20: 3312–3330.
Ischebeck, T., Stenzel, I., Hempel, F., Jin, X., Mosblech, A., and
Heilmann, I. (2011). Phosphatidylinositol-4,5-bisphosphate influences
Nt-Rac5-mediated cell expansion in pollen tubes of Nicotiana tabacum.
Plant J. 65: 453–468.
Ischebeck, T., Vu, L.H., Jin, X., Stenzel, I., Löfke, C., and Heilmann,
I. (2010b). Functional cooperativity of enzymes of phosphoinositide
conversion according to synergistic effects on pectin secretion in
tobacco pollen tubes. Mol. Plant 3: 870–881.
Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1987). GUS
fusions: Beta-glucuronidase as a sensitive and versatile gene fusion
marker in higher plants. EMBO J. 6: 3901–3907.
Kitakura, S., Vanneste, S., Robert, S., Löfke, C., Teichmann, T.,
Tanaka, H., and Friml, J. (2011). Clathrin mediates endocytosis
and polar distribution of PIN auxin transporters in Arabidopsis. Plant
Cell 23: 1920–1931.
Kleine-Vehn, J., Langowski, L., Wisniewska, J., Dhonukshe, P.,
Brewer, P.B., and Friml, J. (2008). Cellular and molecular
requirements for polar PIN targeting and transcytosis in plants. Mol.
Plant 1: 1056–1066.
König, S., Hoffmann, M., Mosblech, A., and Heilmann, I. (2008a).
Determination of content and fatty acid composition of unlabeled
phosphoinositide species by thin-layer chromatography and gas
chromatography. Anal. Biochem. 378: 197–201.
König, S., Ischebeck, T., Lerche, J., Stenzel, I., and Heilmann, I.
(2008b). Salt-stress-induced association of phosphatidylinositol
4,5-bisphosphate with clathrin-coated vesicles in plants. Biochem.
J. 415: 387–399.
Kusano, H., Testerink, C., Vermeer, J.E.M., Tsuge, T., Shimada, H.,
Oka, A., Munnik, T., and Aoyama, T. (2008). The Arabidopsis
Phosphatidylinositol Phosphate 5-Kinase PIP5K3 is a key regulator
of root hair tip growth. Plant Cell 20: 367–380.
Lemmon, M.A., and Ferguson, K.M. (2000). Signal-dependent
membrane targeting by pleckstrin homology (PH) domains. Biochem. J.
350: 1–18.
Markham, J.E., Molino, D., Gissot, L., Bellec, Y., Hématy, K., Marion, J.,
Belcram, K., Palauqui, J.C., Satiat-Jeunemaître, B., and Faure, J.D.
(2011). Sphingolipids containing very-long-chain fatty acids define
a secretory pathway for specific polar plasma membrane protein
targeting in Arabidopsis. Plant Cell 23: 2362–2378.
Matyash, V., Liebisch, G., Kurzchalia, T.V., Shevchenko, A., and
Schwudke, D. (2008). Lipid extraction by methyl-tert-butyl ether for
high-throughput lipidomics. J. Lipid Res. 49: 1137–1146.
Mei, Y., Jia, W.J., Chu, Y.J., and Xue, H.W. (2012). Arabidopsis
phosphatidylinositol monophosphate 5-kinase 2 is involved in root
gravitropism through regulation of polar auxin transport by affecting
the cycling of PIN proteins. Cell Res. 22: 581–597.
Men, S., Boutté, Y., Ikeda, Y., Li, X., Palme, K., Stierhof, Y.D.,
Hartmann, M.A., Moritz, T., and Grebe, M. (2008). Steroldependent endocytosis mediates post-cytokinetic acquisition of
PIN2 auxin efflux carrier polarity. Nat. Cell Biol. 10: 237–244.
Mikami, K., Katagiri, T., Iuchi, S., Yamaguchi-Shinozaki, K., and
Shinozaki, K. (1998). A gene encoding phosphatidylinositol-4phosphate 5-kinase is induced by water stress and abscisic acid in
Arabidopsis thaliana. Plant J. 15: 563–568.
Mueller-Roeber, B., and Pical, C. (2002). Inositol phospholipid
metabolism in Arabidopsis. Characterized and putative isoforms of
18 of 18
The Plant Cell
inositol phospholipid kinase and phosphoinositide-specific phospholipase
C. Plant Physiol. 130: 22–46.
Palme, K., and Gälweiler, L. (1999). PIN-pointing the molecular basis
of auxin transport. Curr. Opin. Plant Biol. 2: 375–381.
Parton, R.M., Fischer-Parton, S., Watahiki, M.K., and Trewavas,
A.J. (2001). Dynamics of the apical vesicle accumulation and the
rate of growth are related in individual pollen tubes. J. Cell Sci. 114:
2685–2695.
Perera, I.Y., Heilmann, I., and Boss, W.F. (1999). Transient and
sustained increases in inositol 1,4,5-trisphosphate precede the
differential growth response in gravistimulated maize pulvini. Proc.
Natl. Acad. Sci. USA 96: 5838–5843.
Perera, I.Y., Heilmann, I., Chang, S.C., Boss, W.F., and Kaufman,
P.B. (2001). A role for inositol 1,4,5-trisphosphate in gravitropic
signaling and the retention of cold-perceived gravistimulation of oat
shoot pulvini. Plant Physiol. 125: 1499–1507.
Perera, I.Y., Davis, A.J., Galanopoulou, D., Im, Y.J., and Boss, W.F.
(2005). Characterization and comparative analysis of Arabidopsis
phosphatidylinositol phosphate 5-kinase 10 reveals differences in
Arabidopsis and human phosphatidylinositol phosphate kinases.
FEBS Lett. 579: 3427–3432.
Perera, I.Y., Hung, C.Y., Brady, S., Muday, G.K., and Boss, W.F.
(2006). A universal role for inositol 1,4,5-trisphosphate-mediated
signaling in plant gravitropism. Plant Physiol. 140: 746–760.
Petersson, S.V., Johansson, A.I., Kowalczyk, M., Makoveychuk,
A., Wang, J.Y., Moritz, T., Grebe, M., Benfey, P.N., Sandberg, G.,
and Ljung, K. (2009). An auxin gradient and maximum in the
Arabidopsis root apex shown by high-resolution cell-specific
analysis of IAA distribution and synthesis. Plant Cell 21: 1659–1668.
Preibisch, S., Saalfeld, S., and Tomancak, P. (2009). Globally
optimal stitching of tiled 3D microscopic image acquisitions.
Bioinformatics 25: 1463–1465.
Rahman, A., Takahashi, M., Shibasaki, K., Wu, S., Inaba, T.,
Tsurumi, S., and Baskin, T.I. (2010). Gravitropism of Arabidopsis
thaliana roots requires the polarization of PIN2 toward the root tip in
meristematic cortical cells. Plant Cell 22: 1762–1776.
Randall, P.J., and Bouma, D. (1973). Zinc deficiency, carbonic
anhydrase, and photosynthesis in leaves of spinach. Plant Physiol.
52: 229–232.
Robert, H.S., and Offringa, R. (2008). Regulation of auxin transport
polarity by AGC kinases. Curr. Opin. Plant Biol. 11: 495–502.
Sauer, M., Paciorek, T., Benková, E., and Friml, J. (2006).
Immunocytochemical techniques for whole-mount in situ protein
localization in plants. Nat. Protoc. 1: 98–103.
Sousa, E., Kost, B., and Malhó, R. (2008). Arabidopsis
phosphatidylinositol-4-monophosphate 5-kinase 4 regulates pollen
tube growth and polarity by modulating membrane recycling. Plant
Cell 20: 3050–3064.
Stenzel, I., Ischebeck, T., König, S., Hołubowska, A., Sporysz, M.,
Hause, B., and Heilmann, I. (2008). The type B phosphatidylinositol-4phosphate 5-kinase 3 is essential for root hair formation in Arabidopsis
thaliana. Plant Cell 20: 124–141.
Stenzel, I., Ischebeck, T., Quint, M., and Heilmann, I. (2012). Variable
regions of PI4P 5-kinases direct PtdIns(4,5)P2 toward alternative
regulatory functions in tobacco pollen tubes. Front. Plant Sci. 2: 114.
Sukumar, P., Edwards, K.S., Rahman, A., Delong, A., and Muday,
G.K. (2009). PINOID kinase regulates root gravitropism through
modulation of PIN2-dependent basipetal auxin transport in Arabidopsis.
Plant Physiol. 150: 722–735.
Tanaka, H., Dhonukshe, P., Brewer, P.B., and Friml, J. (2006).
Spatiotemporal asymmetric auxin distribution: A means to coordinate
plant development. Cell. Mol. Life Sci. 63: 2738–2754.
Thole, J.M., and Nielsen, E. (2008). Phosphoinositides in plants:
Novel functions in membrane trafficking. Curr. Opin. Plant Biol. 11:
620–631.
van Leeuwen, W., Vermeer, J.E., and Gadella, T.W., Jr.,., and Munnik,
T. (2007). Visualization of phosphatidylinositol 4,5-bisphosphate in the
plasma membrane of suspension-cultured tobacco BY-2 cells and
whole Arabidopsis seedlings. Plant J. 52: 1014–1026.
Wang, C., Yan, X., Chen, Q., Jiang, N., Fu, W., Ma, B., Liu, J., Li, C.,
Bednarek, S.Y., and Pan, J. (2013). Clathrin light chains regulate
clathrin-mediated trafficking, auxin signaling, and development in
Arabidopsis. Plant Cell 25: 499–516.
Watson, R.T., Kanzaki, M., and Pessin, J.E. (2004). Regulated
membrane trafficking of the insulin-responsive glucose transporter
4 in adipocytes. Endocr. Rev. 25: 177–204.
Zegzouti, H., Anthony, R.G., Jahchan, N., Bögre, L., and
Christensen, S.K. (2006). Phosphorylation and activation of
PINOID by the phospholipid signaling kinase 3-phosphoinositidedependent protein kinase 1 (PDK1) in Arabidopsis. Proc. Natl. Acad.
Sci. USA 103: 6404–6409.
Zhao, Y., Yan, A., Feijó, J.A., Furutani, M., Takenawa, T., Hwang, I.,
Fu, Y., and Yang, Z. (2010). Phosphoinositides regulate clathrindependent endocytosis at the tip of pollen tubes in Arabidopsis and
tobacco. Plant Cell 22: 4031–4044.
Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L., and Gruissem,
W. (2004). GENEVESTIGATOR. Arabidopsis microarray database
and analysis toolbox. Plant Physiol. 136: 2621–2632.
Phosphatidylinositol 4,5-Bisphosphate Influences PIN Polarization by Controlling
Clathrin-Mediated Membrane Trafficking in Arabidopsis
Till Ischebeck, Stephanie Werner, Praveen Krishnamoorthy, Jennifer Lerche, Mónica Meijón, Irene
Stenzel, Christian Löfke, Theresa Wiessner, Yang Ju Im, Imara Y. Perera, Tim Iven, Ivo Feussner,
Wolfgang Busch, Wendy F. Boss, Thomas Teichmann, Bettina Hause, Staffan Persson and Ingo
Heilmann
Plant Cell; originally published online December 10, 2013;
DOI 10.1105/tpc.113.116582
This information is current as of June 17, 2017
Supplemental Data
/content/suppl/2013/10/21/tpc.113.116582.DC1.html
Permissions
https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X
eTOCs
Sign up for eTOCs at:
http://www.plantcell.org/cgi/alerts/ctmain
CiteTrack Alerts
Sign up for CiteTrack Alerts at:
http://www.plantcell.org/cgi/alerts/ctmain
Subscription Information
Subscription Information for The Plant Cell and Plant Physiology is available at:
http://www.aspb.org/publications/subscriptions.cfm
© American Society of Plant Biologists
ADVANCING THE SCIENCE OF PLANT BIOLOGY