The Type B Phosphatidylinositol-4-Phosphate 5-Kinase 3 IsEssential for Root Hair Formation in Arabidopsis thaliana W

Irene Stenzel,a Till Ischebeck,a Sabine Konig,a Anna Ho1ubowska,a Marta Sporysz,a

Bettina Hause,b and Ingo Heilmanna,1

a Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University Gottingen, 37077

Gottingen, Germanyb Department of Secondary Metabolism, Leibniz Institute for Plant Biochemistry, 06120 Halle (Saale), Germany

Root hairs are extensions of root epidermal cells and a model system for directional tip growth of plant cells. A previously

uncharacterized Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinase gene (PIP5K3) was identified and found to

be expressed in the root cortex, epidermal cells, and root hairs. Recombinant PIP5K3 protein was catalytically active and

converted phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Arabidopsis mutant

plants homozygous for T-DNA–disrupted PIP5K3 alleles were compromised in root hair formation, a phenotype complemented

by expression of wild-type PIP5K3 cDNA under the control of a 1500-bp PIP5K3 promoter fragment. Root hair–specific PIP5K3

overexpression resulted in root hair deformation and loss of cell polarity with increasing accumulation of PIP5K3 transcript.

Using reestablishment of root hair formation in T-DNA mutants as a bioassay for physiological functionality of engineered

PIP5K3 variants, catalytic activity was found to be essential for physiological function, indicating that PtdIns(4,5)P2 formation is

required for root hair development. An N-terminal domain containing membrane occupation and recognition nexus repeats,

which is not required for catalytic activity, was found to be essential for the establishment of root hair growth. Fluorescence-

tagged PIP5K3 localized to the periphery of the apical region of root hair cells, possibly associating with the plasma membrane

and/or exocytotic vesicles. Transient heterologous expression of full-length PIP5K3 in tobacco (Nicotiana tabacum) pollen

tubes increased plasma membrane association of a PtdIns(4,5)P2-specific reporter in these tip-growing cells. The data

demonstrate that root hair development requires PIP5K3-dependent PtdIns(4,5)P2 production in the apical region of root hair

cells.

INTRODUCTION

The uptake of nutrients from the soil into plant roots is facilitated

by the formation of root hairs maximizing the root surface area.

Root hair formation is enhanced by nutrient-limiting conditions,

for instance, in response to limiting iron or inorganic phosphate

(Raghothama, 1999; Gilroy and Jones, 2000; Muller and

Schmidt, 2004), and may enable plants to colonize otherwise

nutrient-restrictive environments. Root hairs form as cytoplasmic

protrusions extending from root epidermal cells (Galway et al.,

1994) and, besides pollen tubes, are a model system for the

study of polar tip growth in plant cells.

The definition of sites of root hair initiation and genetic and cell

biological events driving hair cell elongation has been the focus

of previous studies (Schiefelbein, 2000; Pesch and Hulskamp,

2004; Samaj et al., 2004; Sieberer et al., 2005; Fischer et al.,

2006). The cellular machinery for polar tip growth of root hairs

involves the actin cytoskeleton (Voigt et al., 2005), regulatory

mitogen-activated protein kinases (Samaj et al., 2002), a variety

of regulatory GTP binding proteins (Preuss et al., 2004), and

factors required for vesicle trafficking (Yuen et al., 2005; Song

et al., 2006). Cytoskeletal structures, including microtubules,

help stabilize the elongated hair behind the actively growing tip

region and define the area where the plasma membrane is

expanded and cell wall material is deposited (Sieberer et al.,

2005).

To establish controlled polar tip growth, processes involved in

root hair elongation require tight spatial and temporal regulation.

Although it is not clear how the various elements of the complex

growth machinery are orchestrated, it has been proposed that

the polyphosphoinositide, phosphatidylinositol-4,5-bisphosphate

[PtdIns(4,5)P2], may play a role in the control of root hair growth

(Braun et al., 1999; Vincent et al., 2005). PtdIns(4,5)P2 can affect

multiple physiological processes in all eukaryotic cell types

studied so far by interacting with various protein partners that

are regulated in their biochemical activity or localization (Stevenson

et al., 2000; Mueller-Roeber and Pical, 2002; Meijer and Munnik,

2003; Balla, 2006). Examples for proteins regulated by PtdIns(4,5)P2

include plant ion channels and ATPases (Cote et al., 1996;

Suh and Hille, 2005), plant phospholipase D (Qin et al., 2002), the

actin-modifying enzymes profilin, cofilin, and gelsolin (Drobak

et al., 1994; Lemmon et al., 2002; Doughman et al., 2003;

Wasteneys and Galway, 2003; Wenk and De Camilli, 2004), and

mammalian SNARE complex proteins (Vicogne et al., 2006),

implying a role for PtdIns(4,5)P2 in vesicle fusion (Cremona and

1 Address correspondence to iheilma@uni-goettingen.de.The authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) are: Irene Stenzel(istenze@uni-goettingen.de) and Ingo Heilmann (iheilma@uni-goettingen.de).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.107.052852

The Plant Cell, Vol. 20: 124–141, January 2008, www.plantcell.org ª 2008 American Society of Plant Biologists

De Camilli, 2001; Di Paolo et al., 2004; Gong et al., 2005;

Milosevic et al., 2005). PtdIns(4,5)P2 is also the substrate for

phospholipase C (PLC), yielding diacylglycerol and the soluble

second messenger, inositol 1,4,5-trisphosphate (Berridge, 1983;

Meijer and Munnik, 2003), required for Ca2þ release from internal

stores in both animal (Berridge, 1993) and plant cells (Alexandre

and Lassalles, 1990; Allen et al., 1995; Sanders et al., 2002).

Obviously, regulatory functions described for PtdIns(4,5)P2

include some of those required for a regulator of growth pro-

cesses in tip-growing cells. PtdIns(4,5)P2 has previously been

demonstrated to be involved in tip growth processes in pollen

tubes (Kost et al., 1999; Monteiro et al., 2005; Dowd et al., 2006),

a tip-growing system sharing some components of the growth

machinery with root hairs (Song et al., 2006). In pollen tubes,

Rac-type Rho proteins in the tip plasma membrane physically

associate with an enzyme activity producing PtdIns(4,5)P2 (Kost

et al., 1999), and PtdIns(4,5)P2 has been visualized in a plasma

membrane microdomain of the pollen tube tip (Kost et al., 1999).

As in pollen tubes, the presence of PtdIns(4,5)P2 in the plasma

membrane has previously been demonstrated for the root hair

cell tip (Braun et al., 1999), and phosphoinositides have been

postulated to contribute to the control of root hair development

(Braun et al., 1999; Vincent et al., 2005; Preuss et al., 2006). So

far, however, the enzymes and corresponding genes responsible

for generating PtdIns(4,5)P2 with regulatory function in pollen

tubes or root hairs have not been identified.

This study addresses the role of PtdIns(4,5)P2 formation as a

requirement for root hair growth. PtdIns(4,5)P2 is formed by

phosphorylation of its more abundant precursor lipid, phospha-

tidylinositol-4-phosphate (PtdIns4P), in the D-5 position of the

inositol ring, a reaction catalyzed by PtdIns4P 5-kinases (PI4P

5-kinases) (Drobak et al., 1999; Mueller-Roeber and Pical, 2002).

The Arabidopsis thaliana genome contains 11 genes with simi-

larity to animal PI4P 5-kinases (Mueller-Roeber and Pical, 2002),

only two of which have been experimentally characterized in

detail and been shown to encode enzymes with PI4P 5-kinase

activity in vitro (Elge et al., 2001; Perera et al., 2005). Plant PI4P

5-kinases, including amino acid sequences deduced from puta-

tive and uncharacterized genes, can be classified into types A

and B (Mueller-Roeber and Pical, 2002). The smaller type A

enzymes (isoforms 10 and 11) exhibit a domain structure similar

to that of animal and human PI4P 5-kinases, whereas type B

kinases (isoforms 1 to 9) are unique to plants in containing a large

additional N-terminal extension that includes several membrane

occupation and recognition nexus (MORN) repeats (Mueller-Roeber

and Pical, 2002). MORN repeats are found in various proteins of

both animal and plant origin that mediate protein membrane con-

tacts, such as the Arabidopsis ARC3 protein involved in plastidial

fission (Shimada et al., 2004; Maple et al., 2007) or junctophilins,

which mediate endomembrane-to-plasma membrane attach-

ment in mammalian cells (Takeshima et al., 2000). A recent

report indicates that the N terminus of Arabidopsis PI4P 5-kinase

isoform 1 (PIP5K1) has multiple regulatory effects on enzyme

activity and may guide subcellular localization of the enzyme

(Im et al., 2007). PI4P 5-kinases from different organisms have

been reported to associate with different subcellular locations,

including the plasma membrane of plants, yeast, and mammals

(Perera et al., 1999; Heilmann et al., 2001; Kobayashi et al., 2005;

Santarius et al., 2006), the nucleus of yeast and mammals

(Ciruela et al., 2000; Audhya and Emr, 2003; Santarius et al.,

2006), the actin cytoskeleton of plants, yeast, and mammals

(Desrivieres et al., 1998; Doughman et al., 2003; Davis et al.,

2007), and endomembranes of plants and mammals (Whatmore

et al., 1996; Heilmann et al., 1999; Im et al., 2007).

It has recently been reported that Arabidopsis plants carrying a

T-DNA insertion in the gene encoding the putative PI4P 5-kinase

isoform, PIP5K9, have elevated transcript levels for this gene,

correlated with overall reduced root length (Lou et al., 2007). No

enzymatic activity was demonstrated for a PIP5K9 gene product

in that study, and the restriction of the described phenotype to

roots has not been rationalized in light of the fact that the

disrupted gene is ubiquitously overexpressed throughout the

plant (Lou et al., 2007). Here, we show that a different member of

the Arabidopsis PI4P 5-kinase family, PIP5K3, is expressed in

root epidermal cells and root hairs and that recombinant PIP5K3

is catalytically active as a PI4P 5-kinase in vitro. Both under- and

overexpression of the PIP5K3 gene result in severe disturbance

of root hair directional growth and formation of short or morpho-

logically aberrant root hairs, respectively. The data presented

indicate that the formation of PtdIns(4,5)P2 by PIP5K3 in the

apical region of root hair cells is essential for polar tip growth.

RESULTS

PIP5K3 Is Expressed in Root Epidermis Cells and Root Hairs

In preparation of experiments to define possible physiological

roles of Arabidopsis PI4P 5-kinases, transcript array information

publicly accessible through the Genevestigator portal (Zimmermann

et al., 2004) was reviewed for the tissue-specific expression

patterns of all members of the Arabidopsis PI4P 5-kinase family

(Mueller-Roeber and Pical, 2002). Based on the Genevestigator

data available at the onset of this study, PIP5K3 was identified as

the only Arabidopsis PI4P 5-kinase gene with exclusive expres-

sion in roots, suggesting a specific function in root development

and making PIP5K3 the focus of this investigation. Direct exper-

imental verification of root-specific expression patterns of

PIP5K3 was achieved by RT-PCR (Figure 1A) and by expression

of fusions of a 1500-bp fragment of the 59-untranslated region

upstream of the PIP5K3-coding region with a b-glucuronidase

(GUS) reporter, followed by histochemical staining of transgenic

plant lines for GUS activity (Figures 1B to 1K). The respective

promoter GUS expression patterns were also tested for other

PI4P 5-kinase isoforms suggested by Genevestigator analysis to

be ubiquitously expressed, including in roots, and are described

and discussed in Supplemental Figure 1 and Supplemental Text

and References online). The promoter GUS experiments con-

firmed root-specific expression of the PIP5K3 gene (Figures 1B

and 1C) and revealed that the PIP5K3 promoter is active in cells

of the root cortex and epidermis (Figures 1D to 1F) and in root

hairs (Figure 1G). Only weak or no staining was detected in the

vascular tissue (Figure 1D), as evident especially in root cross

sections (Figures 1E and 1F). No GUS staining was observed with

the PIP5K3 promoter fragment in organs other than roots, as

indicated in Figures 1H to 1K.

PI4P 5-Kinase 3 and Root Hair Development 125

Arabidopsis PI4P 5-Kinase Isoforms Expressed in Roots

Are Active and Catalyze the Conversion of PtdIns4P

to PtdIns(4,5)P2

A prerequisite for further genetic and phenotypic studies is the

characterization of catalytic activity of the PIP5K3 protein or

other putative PI4P 5-kinase isoforms present in roots. Because

the PIP5K3 gene has not yet been functionally characterized,

PIP5K3 was heterologously expressed in Escherichia coli and

the recombinant protein tested for activity in vitro. The catalytic

activity of recombinant PIP5K3 and of some engineered PIP5K3

derivatives relevant to experiments described in Figures 3 to 5, 7,

Figure 1. PIP5K3 Is Expressed in Root Epidermis Cells and Root Hairs.

(A) Abundance of PIP5K3 transcript in different Arabidopsis organs according to RT-PCR analysis using a primer combination amplifying the full-length

PIP5K3 transcript. Flowers and siliques analyzed were taken from 6-week-old plants.

(B) to (K) Histochemical staining for GUS activity in different organs of transgenic plants expressing the GUSplus reporter gene under a 1500-bp

fragment of the PIP5K3 promoter. Samples were stained for GUS activity for 2 h ([C] to [E] and [G]) or for 24 h ([B], [F], and [H] to [K]). All plants shown

were analyzed at an age of 21 d, unless stated otherwise.

(B) Twenty-one-day-old plant.

(C) Two-day-old seedling.

(D) Root close-up. Bar ¼ 100 mm.

(E) and (F) Root cross sections. Bars ¼ 25 mm.

(G) Root hair close-up. Bar ¼ 100 mm.

(H) Flower bud.

(I) Open flower.

(J) Mature leaf.

(K) Stem and siliques. Inset: silique detail with developing seeds.

126 The Plant Cell

and 9 are given in Figure 2. Proteins derived from PIP5K3 that

were also tested (Figure 2) included a truncated PIP5K3 variant

lacking the N-terminal domains (DNT-MORN), a PIP5K3 deriva-

tive with mutated ATP binding site (K442A), a PIP5K3 derivative

mutated in three conserved positions of the catalytic site (S673A/

S680A/S682A; termed TripleA), and a truncated protein encoded

by the T-DNA–disrupted allele pip5k3-4 used for mutant studies

described below (pip5k3-4). Of the PIP5K3-derived proteins

tested, only full-length PIP5K3 and DNT-MORN had catalytic

activity, whereas the activity of the other PIP5K3 derivatives

tested was reduced to the limits of detection (Figure 2B). A minor

activity phosphorylating PtdIns3P to PtdIns(3,4)P2 was observed

for all full-length Arabidopsis PI4P 5-kinases tested, whereas no

activity was detected against PtdIns5P (Table 1). In addition, the

activities of root-expressed PI4P 5-kinase isoforms 1, 2, 7, 8, and

9 with the preferred substrate, PtdIns4P, were tested and are

given in Supplemental Figure 2 online. All proteins were recom-

binantly expressed as fusions to N-terminal maltose binding

protein (MBP) tags. Note that catalytic activity was not precluded

by the N-terminal addition of an MBP tag (Figure 2).

T-DNA Disruption of the PIP5K3 Gene Locus (At2g26420)

Reduces Root Hair Growth

To identify the physiological role of the PIP5K3 gene, a loss-of-

function approach was pursued. A search of the Salk Institute

Genomic Analysis Laboratory collection for T-DNA mutants of

the PIP5K3 gene locus (At2g26420) identified several indepen-

dent T-DNA insertions (Figure 3A) that were screened for homo-

zygosity. Homozygosity was inferred by the inability to amplify

the wild-type allele of PIP5K3 by PCR concomitant with positive

amplification of the T-DNA–tagged allele (see Supplemental

Figure 3 online). Insertion lines SALK_060590 and SALK_001546,

hereafter referred to as pip5k3-1 and pip5k3-2, carry T-DNA

insertions in the 59-untranslated region 248 and 48 bp upstream

of the PIP5K3-coding region, respectively. Insertion lines

SALK_000024 and SALK_026683 carry insertions in the third

intron or the sixth exon, respectively, and will be referred to as

pip5k3-3 and pip5k3-4 in the following text. The positions of the

T-DNA insertions were confirmed by sequencing of the corre-

sponding genomic loci. To test for altered PIP5K3 transcript

abundance in the T-DNA insertion lines, RNA gel blot experi-

ments were performed; however, PIP5K3 transcript levels were

too low to detect. The more sensitive RT-PCR analysis (Figure

3B) and quantitative real-time RT-PCR analysis (Figure 3C)

indicated that the insertion mutants pip5k3-2 and pip5k3-4 had

severely reduced PIP5K3 transcript levels compared with the wild

type, whereas only moderate transcript reduction was observed

in pip5k3-1 and pip5k3-3 plants. Homozygous pip5k3-4-plants

were chosen for a detailed characterization of the T-DNA–

disrupted transcript, and it was established that the correspond-

ing truncated mRNA encoded a truncated protein, pip5k3-4, that

lacks the extreme C terminus, including the activation loop

region (AL) involved in substrate binding (Kunz et al., 2000), and

was catalytically inactive after recombinant expression in E. coli

(Figure 2B).

Correlated to the reduction in PIP5K3 transcript levels (Figure

3C), all homozygous T-DNA insertion mutants exhibited reduced

root hair growth (Figure 4). Compared with wild-type plants

(Figure 4A), the reduction in root hair growth was most severe in

the lines pip5k3-2 (Figure 4C) and pip5k3-4 (Figure 4E) and less

Figure 2. Catalytic Activity of Arabidopsis PIP5K3 and Derived Proteins.

Proteins were recombinantly expressed in E. coli as fusions to N-terminal

MBP tags and were tested for their ability to convert PtdIns4P to

PtdIns(4,5)P2 in vitro.

(A) Schematic representation of PIP5K3 and derived proteins tested. NT,

N terminus (PIP5K31-49); MORN, MORN repeat domain (PIP5K350-227); Lin,

linker domain (PIP5K3228-307); Dim, dimerization domain (PIP5K3308-381);

Cat, catalytic domain (PIP5K3382-705). Arrowheads in K442A and TripleA

indicate mutagenized amino acid positions exchanging a catalytic Lys or

three conserved Ser residues in the catalytic domain for Ala residues,

respectively.

(B) Catalytic activity in recombinant E. coli extracts, as indicated.

Concentrations of recombinant proteins expressed in E. coli were

balanced according to protein gel blot analysis. Data represent the

means of three to four independent experiments 6 SD.

Table 1. Relative Activities of Arabidopsis PI4P 5-Kinase Isoforms

Expressed in Roots

Substrate PtdIns4P/PtdIns3P

Protein PtdIns3P PtdIns4P PtdIns5P Activity Ratio

MBP 2 6 0.3 4 6 0 – –

PIP5K1 5 6 0.8 205 6 17 – 41.0

PIP5K2 52 6 8 1206 6 156 – 23.0

PIP5K3 178 6 27 1972 6 66 – 11.0

PIP5K7 2 6 0.3 37 6 6 – 18.5

PIP5K8 3 6 0.5 27 6 3 – 9.0

PIP5K9 4 6 0.6 142 6 23 – 35.5

Catalytic activities were tested in vitro against different phosphatidy-

linositol-monophosphate substrates and indicate the rate of product for-

mation in fmol min�1. Recombinant protein concentrations were balanced

according to protein gel blot analysis. Data are the means of two inde-

pendent experiments 6 SD.

PI4P 5-Kinase 3 and Root Hair Development 127

pronounced in pip5k3-1 (Figure 4B) and pip5k3-3 (Figure 4D), as

summarized in Figure 4F.

Root Hair Development Is Reestablished in PIP5K3 T-DNA

Insertion Mutants Expressing Full-Length PIP5K3

The observation that the loss of PIP5K3-transcript was corre-

lated with reduced root hair growth (Figures 3 and 4) was

unexpected because the expression of five other active PI4P

5-kinase isoforms in roots (see Supplemental Figures 1 and 2

online) had suggested some functional redundancy. To verify

that the observed reduction in root hair growth was indeed due to

the disruption of the PIP5K3 gene, complementation of the

pip5k3-4 mutant phenotype (Figures 4E and 4F) was attempted

using full-length PIP5K3 or various PIP5K3 derivatives, as indi-

cated (Figure 5). The complementation constructs contained the

respective cDNA clones behind the 1500-bp PIP5K3 promoter

fragment used for the GUS expression experiments shown in

Figure 1 and encoded the respective proteins as fusions to

N-terminal enhanced yellow fluorescent protein (EYFP) tags.

Complementation was evaluated in comparison to nontrans-

formed wild-type plants (Figure 5A) and to pip5k3-4 mutants

(Figure 5B) grown in parallel. The genotypes of the plant lines

used and the presence of correctly sized transcripts correspond-

ing to the ectopically introduced PIP5K3 constructs were deter-

mined by PCR and RT-PCR, respectively (Figure 5G). Expression

of PIP5K3 in the pip5k3-4 mutant resulted in reestablishment of

normal root hair growth (Figure 5C), and root hairs were at an

average even longer than those of wild-type controls (Figure 5H).

Using the reestablishment of root hair growth in the pip5k3-4

mutant (cf. Figure 3) as a bioassay, the functionality of the

PIP5K3-derived proteins was tested. The truncated DNT-MORN

or the inactive K442A or TripleA proteins did not reestablish root

hair formation in the pip5k3-4 background (Figures 5D to 5F), as

summarized in Figure 5H. Root hair stubs forming with expres-

sion of K442A (Figure 5E) appeared thinner than those seen in

other transgenics. As the levels of the introduced proteins in root

hairs were too low to be detected by EYFP fluorescence or by

immunoblotting, expression of the transgenes was verified by

monitoring transcript levels for the various PIP5K3 derivatives

by quantitative real-time RT-PCR (Figure 5I), indicating that the

expression levels of the EYFP-tagged PIP5K3 transgenes intro-

duced into the pip5k3-4 mutant did not exceed the level of

PIP5K3 expression in wild-type plants by more than an average

of ;30%.

Aberrant Root Hair Growth and Morphology with

Overproduction of PIP5K3

The underexpression and complementation data for the PIP5K3

gene (Figures 4 and 5, respectively) indicated that PIP5K3 was an

essential regulator of root hair development and led us to ask

whether root hair formation would also be disturbed by overpro-

duction of EYFP-tagged PIP5K3. The stronger root hair–specific

promoter of Arabidopsis expansin 7 (EXP7) (Cho and Cosgrove,

2002) was used to drive ectopic overexpression of PIP5K3 in root

hairs of wild-type Arabidopsis plants. Compared with nontrans-

formed wild-type plants (Figure 6A), increasing degrees of over-

expression (Figures 6B to 6D) resulted in increasingly deformed

root hairs. From the highly polar appearance in wild-type plants

(Figure 6E) root hair morphology was altered with increasing

PIP5K3 overexpression toward thicker hairs (Figure 6F), curling

hairs (Figure 6G), and, with the highest degree of overexpression,

globular structures (Figure 6H), indicating a gradual loss of

cellular polarity. Increased PIP5K3 transcript levels in the over-

expressor lines were documented independently by quantitative

Figure 3. T-DNA Disruption of the PIP5K3 Gene Locus (At2g26420)

Results in Reduced PIP5K3 Transcript Levels.

(A) Structure of the genomic PIP5K3 locus with positions of the four

T-DNA insertions, pip5k3-1, pip5k3-2, pip5k3-3, and pip5k3-4. Arrows

indicate the position of primers used for RT-PCR analysis of transcript

levels.

(B) Abundance of full-length PIP5K3 transcript in roots of 3- to 4-week-

old plants homozygous for the respective T-DNA insertions, as deter-

mined by RT-PCR. Note that a pip5k3-4 protein would be truncated at the

extreme C terminus and catalytically inactive (Figure 2B). ACT8, control.

(C) Real-time RT-PCR analysis of PIP5K3 transcript levels in roots of

3- to 4-week-old wild-type or mutant plants, as indicated, using a primer

combination amplifying a 64-bp fragment of the cDNA stretch encoding

the catalytic region of the PIP5K3 protein. Dashed line, wild-type PIP5K3

transcript levels. Real-time RT-PCR data are given relative to transcript

levels detected in wild-type plants and represent the mean 6 SE of two

independent experiments.

128 The Plant Cell

real-time RT-PCR (Figure 6I) and by RT-PCR (Figure 6I, inset).

Data shown are from representative transgenic lines. Morpho-

logical changes as those reported were found to correlate with

transcript accumulation in eight of eight independent transgenic

lines.

Effects of Ectopic Expression of K442A and Truncated

DNT-MORN on Root Hair Morphology

Because under- and overexpression of PIP5K3 both affected

root hair morphology, the effects of overexpressing the inactive

K442A protein or the truncated DNT-MORN protein were tested.

Figure 4. Reduced Root Hair Growth of T-DNA Insertion Mutants of PIP5K3.

Plants were grown on agar plates for 8 d, and digital images were taken.

(A) to (E) Representative root hair phenotypes. Bars ¼ 100 mm.

(A) Wild-type plants.

(B) pip5k3-1 mutant.

(C) pip5k3-2 mutant.

(D) pip5k3-3 mutant.

(E) pip5k3-4 mutant.

(F) Quantification of root hair length in the wild type and in plants carrying T-DNA insertions, as indicated. The asterisks indicate significantly reduced

root hair length compared with wild-type controls according to a Student’s t test (*, P < 0.05; **, P < 0.01; n > 300).

PI4P 5-Kinase 3 and Root Hair Development 129

Figure 5. Root Hair Phenotypes of pip5k3-4 Plants Ectopically Expressing PIP5K3 or Derived Proteins.

Complementation of the pip5k3-4 mutant phenotype was tested by ectopic expression of PIP5K3 or various derived proteins under a 1500-bp fragment

of the intrinsic PIP5K3 promoter in the pip5k3-4 background.

(A) and (B) Root hair phenotypes of wild-type plants (A) and pip5k3-4 mutant plants (B) grown in parallel.

(C) to (F) Root hair phenotypes of pip5k3-4 plants with expression of PIP5K3 (C), DNT-MORN (PIPK3228-705; [D]); K442A (E), and TripleA (F). Bars¼ 200

mm. Images are representative for results obtained with at least five independent transgenic lines.

(G) The presence of the T-DNA insertions was shown by PCR using genomic DNA as a template and a combination of primers specific for the T-DNA

130 The Plant Cell

Both proteins were expressed as fusions to the fluorescence tag

RedStar. Root hairs of nontransformed wild-type plants (Figure

7A) and of plants expressing RedStar alone (Figure 7B) did not

differ in length. The introduction of RedStar-tagged K442A under

the EXP7 promoter resulted in root hair deformation and branch-

ing (Figure 7C). Overexpression of RedStar-tagged DNT-MORN

protein (Figure 7D) resulted in a phenotype similar to that ob-

served with K442A. Note that DNT-MORN was catalytically

active (Figure 2B) but lacks an N-terminal domain of unknown

physiological role. Phenotypes were observed in eight out of

eight (K442A) and six out of six (DNT-MORN) transgenic lines;

however, root hairs were never globally altered, and all plants

also had root areas with wild-type-like root hairs. The presence

or absence of K442A and DNT-MORN transcripts of the correct

sizes was verified by RT-PCR (Figure 7E). The PIP5K3 expres-

sion levels in the control lines and the overexpressors were

determined by quantitative real-time RT-PCR (Figure 7F) and

were found to be substantially increased over wild-type PIP5K3

transcript levels. Note that the real-time RT-PCR does not

distinguish between the wild-type allele and the mutated or

truncated variant of the PIP5K3 gene. The data are consistent

with dominant-negative effects resulting from overexpression of

K442A or DNT-MORN.

PIP5K3 Localizes to the Apical Region of Root Hair Cells

Because enzymes controlling cell polarity and polar growth

distribute to their subcellular sites of action, the subcellular

localization of PIP5K3 in root hairs was investigated by confocal

laser scanning microscopy. An EYFP tag was N-terminally linked

to PIP5K3, and the fusion protein was expressed under the EXP7

promoter in wild-type Arabidopsis plants. The resulting distribu-

tion of EYFP fluorescence is shown in Figure 8. Confocal images

of the bright-field (Figure 8A) and the EYFP channel (Figure 8B)

indicating PIP5K3 localization were synchronously recorded. A

merge of these images is given in Figure 8C. The distribution of

EYFP fluorescence indicates that the EYFP-PIP5K3 fusion pro-

tein is restricted to the periphery of the apical region of root hair

cells. In root hairs expressing EYFP-PIP5K3 under the control of

the EXP7 promoter, the EYFP-PIP5K3 fluorescence did not show

a tight distribution typical for plasma membrane proteins. While

formation of PtdIns(4,5)P2 was expected to occur at the plasma

membrane (Braun et al., 1999; Kost et al., 1999; Perera et al.,

1999; Heilmann et al., 2001; Kobayashi et al., 2005; van Leeuwen

et al., 2007), EYFP-PIP5K3 also decorated a conical pattern of

small cytosolic particles in the apex of root hair cells in addition to

possible plasma membrane association (Figure 8D). To docu-

ment localization of EYFP fluorescence in relation to root hair

autofluorescence, a confocal cross section of 1.6 mm thickness

close to the apical tip of a root hair was scanned, indicating a ring

of EYFP fluorescence immediately inside the autofluorescing cell

wall (Figure 8E). The distribution of EYFP-PIP5K3 fluorescence

reported was only observed in growing root hairs close to the

root tip, such as that shown in Figure 8, whereas older root hairs

exhibited only autofluorescence.

Expression of PIP5K3 Increases Plasma Membrane Levels

of PtdIns(4,5)P2

To determine whether plasma membrane PtdIns(4,5)P2 levels

were influenced by the expression of PIP5K3 in tip-growing plant

cells, PIP5K3 and K442A were each transiently expressed in

tobacco (Nicotiana tabacum) pollen tubes, together with the

RedStar-tagged Pleckstrin homology (PH) domain of the human

PLCd1 (HsPLCd1-PH domain), which specifically recognizes

PtdIns(4,5)P2 (Varnai and Balla, 1998) (Figure 9). Pollen tubes

were chosen instead of root hairs because the system is similar

to root hairs in many respects, it can easily be transiently trans-

formed by particle bombardment, and its autofluorescence is

negligible. The active and inactive proteins both localized to the

apical plasma membrane of the pollen tube tip in a pattern

resembling the distribution of EYFP-PIP5K3 in root hair cells (see

Supplemental Figure 4 online; compare Figures 8B and 8D). The

HsPLCd1-PH domain reporter (Figures 9A and 9B, middle pan-

els) also localized to the apical plasma membrane of the pollen

tubes, as previously reported (Kost et al., 1999; Dowd et al.,

2006). The right panel in Figure 9A illustrates roughly equal

intensities of PIP5K3 and HsPLCd1-PH domain, resulting in a

yellow color on the merged image, whereas unequal intensities of

K442A and HsPLCd1-PH domain shown in Figure 9B result in

green plasma membrane fluorescence of the merged image.

When fluorescence intensities were quantified (Figure 9C), the

fluorescence distribution of RedStar-HsPLCd1-PH with coex-

pression of active PIP5K3 was clearly plasma membrane associ-

ated (Figure 9C, top right panel), whereas equivalent expression of

the inactive K442A resulted in a more diffuse distribution of the

PtdIns(4,5)P2 reporter (Figure 9C, bottom right panel), indicating

that a higher proportion of the reporter remained in the cyto-

plasm. While synchronous imaging of PIP5K3-derived proteins

Figure 5. (continued).

insert (sense) and the PIP5K3 gene (antisense). The presence of correctly sized transcripts corresponding to the ectopic transgenes introduced was

tested by RT-PCR on root RNA, as indicated, using a primer combination specific for the cDNA stretch encoding the N-terminal EYFP tag (sense) and

for that encoding the extreme C terminus of the PIP5K3 protein (antisense). ACT8 served as an RNA loading control for the RT-PCR experiment. All

plants were analyzed 8 d after germination.

(H) Quantification of root hair lengths in 8-d-old wild type and transgenics. EYFP, control expressing EYFP alone under the PIP5K3 promoter fragment.

Asterisks indicate significantly increased root hair length compared with those of the pip5k3-4 background according to a Student’s t test (**, P < 0.01;

n > 300).

(I) Real-time RT-PCR analysis of PIP5K3 transcript levels in roots of wild-type, mutant, and complemented mutant plants, as indicated. The primer

combination used amplified a 64-bp fragment of the cDNA stretch encoding the catalytic region of the PIP5K3 protein. Real-time RT-PCR data are given

relative to transcript levels detected in wild-type plants and represent the mean 6 SE of two independent experiments.

PI4P 5-Kinase 3 and Root Hair Development 131

with the HsPLCd1-PH domain was successful in pollen tubes

(Figure 9), no interpretable images were obtained in correspond-

ing experiments on root hairs because of interfering autofluo-

rescence at the low HsPLCd1-PH expression levels required for

meaningful interpretation.

DISCUSSION

The data presented indicate an essential role for the PI4P

5-kinase PIP5K3 in the development of root hairs in Arabidopsis.

The requirement of PIP5K3 for root hair formation must be

reviewed in light of its catalytic activity and its capability to

generate PtdIns(4,5)P2 (Figure 2). The in vitro activity of recom-

binant PIP5K3 protein indicates a clear preference for the con-

version of PtdIns4P to PtdIns(4,5)P2 (Table 1), which is consistent

with the presence of a conserved Glu residue (Glu-668) in the AL

of PIP5K3 implicated in determining PI4P 5-kinase specificity

(Kunz et al., 2000). A low but detectable activity of PIP5K3 and

other isoforms against PtdIns3P (Table 1) is consistent with side

activities previously reported for PIP5K1 (Elge et al., 2001) and

PIP5K10 (Perera et al., 2005), which also exhibit minor activity

with this substrate. For the ubiquitous PIP5K1, enhanced activity

Figure 6. Aberrant Root Hair Morphology with Overproduction of PIP5K3.

The wild-type PIP5K3 allele was expressed under the root-hair-specific EXP7 promoter in wild-type plants.

(A) Root hair morphology of wild-type controls grown in parallel.

(B) to (D) Overexpressor lines shown were chosen to document morphological alteations with increasing levels of PIP5K3 transcript.

(E) to (H) Magnifications of root hairs exhibiting morphologies characteristic for wild-type plants (E) or for plants with increasing levels of PIP5K3

expression ([F] to [H]). Bars ¼ 200 mm in (A) to (D) and 100 mm (E) to (H).

(I) PIP5K3 transcript accumulation was monitored independently by real-time RT-PCR, using a primer combination amplifying a 64-bp fragment of

PIP5K3 as described above and by RT-PCR-analysis (inset) using a primer combination amplifying the full-length PIP5K3 transcript. Real-time RT-PCR

data are given relative to transcript levels detected in wild-type plants and represent the mean 6 SE of two independent experiments. PIP5K3, transcript

levels; ACT8, control. A to D refer to panels above. All plants were analyzed 8 d after germination. Altered root hair morphology correlated to PIP5K3

expression levels was observed in eight out of eight transgenic lines analyzed.

132 The Plant Cell

was reported with removal of the MORN domain of that protein,

indicating an autoinhibitory function for the MORN domain (Im

et al., 2007). In our in vitro activity tests with the PIP5K3-derived

DNT-MORN protein, no such enhancement of PI4P 5-kinase

activity was observed (Figure 2B). Possible explanations include

that autoinhibitory effects previously described (Im et al., 2007)

were specific for PIP5K1 or that in this study the MBP tag

N-terminally attached to the MORN domain of PIP5K3 interfered

with its regulatory functions. The latter scenario appears unlikely,

however, in light of the observation that in vivo functionality

of PIP5K3 was given with expression of PIP5K3 fused to an

N-terminal EYFP reporter (Figure 5C), which is roughly equal in

size and bulk to the MBP tag. The activity tests demonstrate the

importance of the extreme C-terminal end of the PIP5K3 protein,

including the AL, for catalytic activity because the truncated

pip5k3-4 protein was not active (Figure 2B). The loss of catalytic

activity with exchange of three conserved Ser residues in the

TripleA mutant (Figure 2B) attributes importance to the extreme

C-terminal portion of the PIP5K3 protein and suggests PIP5K3

positions Ser-673, Ser-680, and Ser-682 as candidate sites for

posttranslational regulation.

The results of in vitro biochemical characterization of PIP5K3

and its engineered variants (Figure 2) were in congruence with

evidence from genetic studies and complementation tests. In-

dependent T-DNA insertion mutants for the PIP5K3 gene, fore-

most those homozygous for alleles pip5k3-2 and pip5k3-4, were

compromised in root hair development (Figure 4), indicating that

the observed effects on root hair growth were due to altered

expression of PIP5K3, rather than to additional T-DNA insertions

elsewhere in the genome. This notion is further supported by the

fact that PIP5K3 T-DNA insertion mutants ectopically expressing

the wild-type allele of PIP5K3 under its intrinsic promoter ex-

hibited wild-type-like root hair growth (Figure 5C) and that

overexpression of the inactive K442A exerted subtle but repro-

ducible dominant-negative effects on root hair growth (Figure

7C). The observation that elimination of a single gene encoding

one of 11 PI4P 5-kinases (Figures 3 and 4), six of which are

expressed in roots (shown for PIP5K2 and PIP5K9 in Supple-

mental Figure 1 online) and are all catalytically active (Table 1;

see Supplemental Figure 2 online), nonetheless resulted in a

severe phenotype indicates that other PI4P 5-kinases, such as

PIP5K9 also expressed in root hairs (see Supplemental Figure

1 online), did not functionally compensate for the loss of PIP5K3

expression in regard to root hair development. This result is

consistent with results presented in Figure 2 and Supplemental

Figure 7. Effects of K442A or DNT-MORN Expression on Root Hair

Growth.

(A) to (D) The inactive PIP5K3-derived K442A protein or the truncated

DNT-MORN protein was expressed in wild-type plants as fusions to

N-terminal RedStar tags under the control of the EXP7 promoter. Images

were taken from 8-d-old plants; the transgenic lines are representative

for eight (K442A) and six (DNT-MORN) independent lines tested. To

varying degrees, all lines also exhibited regions with unaltered root hairs.

Bars ¼ 200 mm.

(A) Wild-type control.

(B) RedStar control.

(C) and (D) Root hair morphology observed with expression of K442A (C)

or DNT-MORN (D).

(E) RT-PCR detection of correctly sized ectopic PIP5K3 transcripts for

the constructs introduced, as indicated, using a primer combination

specific for the cDNA encoding the RedStar tag (sense) and for that of the

extreme C terminus of the PIP5K3-derived expressed proteins (anti-

sense). RedStar, RedStar transcript, as amplified with RedStar-specific

primers; ACT8, actin control.

(F) Real-time RT-PCR analysis of PIP5K3 transcript levels in plant lines,

as indicated, using a primer combination for the 64-bp PIP5K3 fragment

described above. Dashed line, wild-type PIP5K3 transcript level. Real-

time RT-PCR data are given relative to transcript levels detected in

wild-type plants and represent the mean 6 SE of two independent ex-

periments. Note that the real-time RT-PCR does not distinguish between

wild-type and mutated alleles of the PIP5K3 gene. All plants were

analyzed 8 d after germination.

PI4P 5-Kinase 3 and Root Hair Development 133

Figure 2 online, indicating that recombinant PIP5K3 is by far the

most catalytically active PI4P 5-kinase of the isoforms expressed

in roots. It is interesting to note that the pip5k3-1 mutant carrying

a T-DNA insertion only 248 bp upstream of the coding region was

only moderately impaired in PIP5K3 transcript accumulation

(Figure 3C) and also only moderately impaired in root hair growth

(Figure 4B), suggesting that the 248-bp region still in place may

contain most necessary regulatory elements of the PIP5K3

promoter. It was also a surprise to observe that the pip5k3-3

mutant exhibited substantial PIP5K3 transcript levels and that

the T-DNA insertion in intron three did not prevent efficient mRNA

splicing (Figure 3). From the phenotypes exhibited by the mutant

lines pip5k3-2 and pip5k3-4, which had strongly reduced PIP5K3

transcript levels (Figure 3C), it is clear that PIP5K3 has an

important function in the control of root hair growth.

To better understand the function of PIP5K3 in root hair

formation, inactive or truncated variants of PIP5K3 were intro-

duced into the pip5k3-4 mutant background (Figure 5), and the

capability of these constructs to reestablish root hair formation

was statistically evaluated (Figure 5H). While ectopic expression

of the wild-type allele of the PIP5K3 gene resulted in full com-

plementation of the pip5k3-4 phenotype (Figure 5C), catalytically

inactive K442A (Figure 5E) and TripleA (Figure 5F) did not

reestablish root hair formation in the pip5k3-4 background.

Whereas PIP5K3 protein levels in root hairs were too low to be

immunodetected, the following arguments indicate that the key

truncations used in this experiment, K442A and DNT-MORN,

were introduced into plants as functional proteins: (1) pheno-

types observed with expression of PIP5K3 and K442A contrasted

radically (cf. Figures 5C and 5E), although the proteins differed

only in one amino acid position not likely to result in a dramatic

difference in protein stability; (2) overexpression of DNT-MORN

under the EXP7 promoter in wild-type plants resulted in de-

formed root hairs (Figure 7D), indicating that the DNT-MORN

protein was functional in those plants and likely not instable. The

combined results indicate that catalytic activity and, thus,

PtdIns(4,5)P2 production are required for root hair formation.

The phenotype with TripleA expression also supports the notion

that a truncated pip5k3-4 transcript does not encode a functional

protein, which correlates well with the observed phenotype of the

pip5k3-4 mutant (Figure 4E). It is interesting to note that expres-

sion of DNT-MORN did not reestablish root hair formation (Figure

5D), despite full catalytic activity in vitro (Figure 2), leading us to

conclude that PIP5K3 functionality in root hair development

requires additional factors other than catalytic activity alone.

Because PtdIns(4,5)P2 has previously been described to form

a lipid microdomain in the plasma membrane of root hair tips

(Braun et al., 1999) and pollen tubes (Kost et al., 1999) and to be

generally plasma membrane associated in plant cells (van

Leeuwen et al., 2007), the plasma membrane was also an

expected localization for PIP5K3. The subcellular distribution of

overexpressed EYFP-tagged PIP5K3 suggested that the en-

zyme localizes to the extreme periphery of the apical tip region of

root hairs (Figure 8) and, with heterologous expression, also of

pollen tubes (see Supplemental Figure 4 online). In addition to a

likely plasma membrane association, EYFP-PIP5K3 also deco-

rated cytosolic particles (Figure 8D) in a pattern resembling the

distribution of exocytotic vesicles in pollen tubes (Lancelle and

Hepler, 1992; Parton et al., 2001). The fluorescence distribution

of EYFP-PIP5K3 was imaged in wild-type plants expressing the

fusion protein under the control of the EXP7 promoter fragment

also used for overexpression studies. No clear results were

obtained using a weaker intrinsic PIP5K3 promoter fragment.

Therefore, it is not clear whether the natural subcellular distribu-

tion of PIP5K3 in the root hair tip may be restricted to the plasma

membrane, to vesicles, or to both. Another possible localization

is cortical actin cytoskeletal structures. It is possible that PIP5K3

localization can change dynamically between different mem-

branes or nonmembrane locations, as has been reported previ-

ously for PIP5K1 (Im et al., 2007). The determinants of subcellular

localization of PIP5K3 thus remain a subject for future studies.

As results so far indicated that PIP5K3 was an active PI4P

5-kinase possibly localized to the plasma membrane or to

vesicles in the apical region of growing root hair cells, we tested

whether expression of PIP5K3 in pollen tubes would increase

plasma membrane levels of PtdIns(4,5)P2 (Figure 9). When

PIP5K3 and the inactive K442A were each coexpressed with

the PtdIns(4,5)P2 reporter HsPLCd1-PH, expression of the active

enzyme resulted in greatly increased plasma membrane asso-

ciation of reporter fluorescence (Figure 9C), indicating increased

levels of PtdIns(4,5)P2, consistent with our expectation based on

the localization data (Figure 8) and previous data on the topic

Figure 8. Subcellular Localization of PIP5K3 in Root Hairs.

EYFP-tagged PIP5K3 was expressed in wild-type plants under the

control of the EXP7 promoter, and the fluorescence distribution was

monitored in 8-d-old plants by confocal laser scanning microscopy.

Bright-field and fluorescence channels were imaged synchronously.

Images shown are representative for a growing root hair with vivid

cytoplasmic streaming expressing EYFP-PIP5K3 under the EXP7 pro-

moter. Bars ¼ 20 mm.

(A) Bright-field image.

(B) EYFP-PIP5K3 fluorescence.

(C) Merge of images (A) and (B).

(D) Magnified view of a 1.0-mm confocal section through the root hair

apex. The arrowhead indicates a cone-shaped distribution of EYFP-

PIP5K3–decorated cytosolic particles close to the apex of the root hair

cell.

(E) Confocal section (1.6 mm) through the apical region of a root hair, as

indicated by the dashed line in (C). Left panel, cell wall autofluorescence;

middle panel, EYFP-PIP5K3 fluorescence; right panel, merged image.

134 The Plant Cell

(Braun et al., 1999; Kost et al., 1999; Perera et al., 1999; Heilmann

et al., 2001; Dowd et al., 2006; van Leeuwen et al., 2007). By

contrast, plasma membrane fluorescence of the PtdIns(4,5)P2

reporter was much reduced when coexpressed with the inactive

K442A (Figure 9B). A low HsPLCd1-PH background signal

in pollen tubes expressing K442A can be attributed to the

presence of PtdIns(4,5)P2 formed by pollen tube endogenous

PI4P 5-kinases. While corresponding experiments with root hairs

were not successful because of high root hair autofluorescence,

the results from the heterologous pollen tube system (Figure 9)

correspond well with the in vitro characterization of PIP5K3 and

K442A (Figure 2) and with the effects of the two proteins in the

mutant complementation tests (Figure 5). Overall, the results

presented are consistent with the hypothesis that PIP5K3 pro-

duces PtdIns(4,5)P2 in the tips of growing root hair cells that is

required for growth.

A number of reported functions of PtdIns(4,5)P2 can be

envisioned to take part in the control of directional tip growth in

the apex of growing root hairs, as has been outlined in the

Introduction. The loss of cellular polarity observed in Arabidopsis

root hairs overexpressing PIP5K3 (Figure 6) is similar to pheno-

types observed with increased PtdIns(4,5)P2 levels in pollen

tubes due to inactivation of PLC (Dowd et al., 2006; Helling et al.,

2006), and pollen tubes strongly expressing the EYFP-PIP5K3

fusions also exhibited tip swelling and a loss of polar tip growth.

The observation that expression of PIP5K3 in the pip5k3-4

background resulted in nondeformed root hairs that were slightly

longer than those of wild-type plants (Figure 5G), whereas

overexpression resulted in severe deformation (Figure 6), sug-

gests that a fine balance of PIP5K3 expression may be required

for normal root hair development.

In analogy to an animal model for the involvement of

PtdIns(4,5)P2 in synaptic exocytosis (Cremona and De Camilli,

2001), a possible explanation for reduced root hair growth in the

absence of PIP5K3 is suggested by the notion that in mammalian

cells the assembly of the protein machinery required for the

fusion of PtdIns4P-coated vesicles with the plasma membrane

depends on the formation of PtdIns(4,5)P2 (Di Paolo et al., 2004;

Gong et al., 2005; Milosevic et al., 2005; Vicogne et al., 2006).

Compromised vesicle trafficking from the endoplasmic reticulum

to the Golgi due to continuous inactivation of the monomeric

GTPase, ARF1, in Arabidopsis plants has been shown to result in

reduced growth of root hairs and pollen tubes (Song et al., 2006),

similar to that seen in this study with disruption of the PIP5K3

gene. In combination with the reported accumulation of PtdIns4P-

coated exocytotic vesicles in the tip of growing root hair cells

(Preuss et al., 2006) the results presented suggest that PIP5K3

may control root hair elongation by providing PtdIns(4,5)P2 re-

quired for vesicle-to-plasma membrane fusion in the apex of the

hair cell, either at the surface of the plasma membrane target area

or that of the fusing vesicle.

A possible alternative mechanism by which plasma membrane–

associated PIP5K3 may affect root hair elongation relates to the

control of F-actin dynamics in analogy to the situation in yeast,

where the only PI4P 5-kinase, MSS4p, mediates the attachment

of F-actin to the plasma membrane and is required for yeast

budding (Desrivieres et al., 1998). In analogy to the yeast model,

PIP5K3 may yield PtdIns(4,5)P2 controlling F-actin-modulating

Figure 9. Expression of PIP5K3 in Pollen Tubes Increases Plasma

Membrane Levels of PtdIns(4,5)P2.

(A) and (B) EYFP-PIP5K3 and the catalytically inactive EYFP-K442A

protein were each coexpressed in tobacco pollen tubes with the Red-

Star-tagged HsPLCd1-PH, which specifically binds to PtdIns(4,5)P2.

EYFP and RedStar fluorescence (left and middle panels, respectively)

were synchronously recorded by confocal laser scanning microscopy

after incubation of transformed pollen for 10 h. Right panels, merged

images. Yellow color indicates colocalization of EYFP and RedStar

signals of equal intensity. Bars ¼ 10 mm.

(A) PIP5K3.

(B) K442A.

(C) Quantification of relative fluorescence intensities in horizontal sec-

tions indicated in (A) and (B). Fluorescence intensities were normalized

against the highest value for each scan, set as 1. Transient expression

was performed under identical conditions. Images were not individually

adjusted for brightness or contrast. Data presented are from a repre-

sentative experiment. Of 36 transformed pollen tubes observed for

PIP5K3 or K442A, 18 and 19 tubes, respectively, exhibited the pattern

shown. Expression levels of the remaining tubes were either too low to

visualize any RedStar fluorescence with expression of K442A or were too

high for meaningful imaging.

PI4P 5-Kinase 3 and Root Hair Development 135

proteins, such as profilin or gelsolin, that may be associated with

F-actin strands binding to PIP5K3 via its Lin domain (Davis et al.,

2007). A lack of PIP5K3 in root hairs of the Arabidopsis mutants

described may reduce directional membrane traffic by prevent-

ing F-actin polymerization at the plasma membrane.

In summary, the data presented indicate PIP5K3 as a func-

tional component of the machinery controlling directional tip

growth of root hairs. The PIP5K3 protein catalyzes the produc-

tion of PtdIns(4,5)P2 in the apical region of root hair cells and may

control vesicle trafficking and/or cytoskeletal structures required

for root hair growth. The identification of the PI4P 5-kinase

responsible for the production of PtdIns(4,5)P2 involved in root

hair growth provides a key tool needed to determine the partic-

ular physiological processes in the root hair that are controlled by

that lipid. Future work will be directed toward elucidating the

exact roles of PtdIns(4,5)P2 in the cell biology of root hair

formation.

METHODS

Plant Growth Conditions

All experiments were performed with Arabidopsis thaliana ecotype

Columbia-0 (Col-0). Seeds were surface-sterilized for 5 min in 75%

ethanol, followed by 20 min incubation in 6% (w/v) NaOCl in 0.1% (w/v)

Triton X-100, and washed five times in sterile distilled water. Before

plating, seeds were vernalized at 48C for 2 d, followed by culture on half-

strength Murashige and Skoog (MS) medium including basal salt mixture

(Duchefa) containing 1% (w/v) sucrose and 0.7% (w/v) agar (Duchefa) at

228C under continuous light.

Primers

Sequences for all primers named below are provided in Supplemental

Table 1 online.

Genotyping of T-DNA Insertion Lines

T-DNA insertion lines (pip5k3-1, SALK_060590; pip5k3-2, SALK_001546;

pip5k3-3, SALK_000024; and pip5k3-4, SALK_026683) in the Arabidopsis

Col-0 background were obtained from the Nottingham Arabidopsis Stock

Centre. A PCR-based approach was used to identify homozygous lines

and to confirm the exact insertion position of the T-DNA. For the analysis

of the pip5k3-1 mutant and pip5k3-2 mutant, primers used were LBa1,

SALK_060590_reverse, and SALK_060590_forw. For the analysis of the

pip5k3-3 mutant, primers used were LBa1, SALK_00024_reverse, and

SALK_000024_forw. For the analysis of the pip5k3-4 mutant, primers

used were LBa1, SALK_026683_reverse, and SALK_026683_forw.

Cloning of cDNA or Genomic Constructs

PI4P 5-Kinase Coding Sequences

The cDNA sequences of Arabidopsis PI4P 5-kinase genes PIP5K1, PIP5K2,

PIP5K7, PIP5K8, and PIP5K9 were amplified from cDNA prepared from

young Arabidopsis inflorescences using the following primer combinations:

PIP5K1, PIP5K1Nco_for/PIP5K1Nco_rev; PIP5K2, PIP5K2Pci_for/PIP5K2P-

ci_rev; PIP5K7, PIP5K7Nco_for/PIP5K7Nco_rev; PIP5K8, PIP5K8Nco_for/

PIP5K8Nco_rev; and PIP5K9, PIP5K9Nco_for/PIP5K9Nco_rev. The cDNA of

PIP5K3 was isolated by RT-PCR from cDNA of 14-d-old roots grown on agar

plates using the primer combination PIP5K3Nco_for/PIP5K3Nco_rev. PCR-

fragments were subcloned into the vector pGEM-Teasy (Promega) and

sequenced. The resulting plasmids were designated as PIP5K1-pGEM-

Teasy, PIP5K2-pGEM-Teasy, PIP5K3-pGEM-Teasy, PIP5K7-pGEM-Teasy,

PIP5K8-pGEM-Teasy, and PIP5K9-pGEM-Teasy, respectively.

Mutagenesis of the PIP5K3 Coding Sequence

To obtain the cDNA clone encoding K442A, the PIP5K3 coding sequence

was altered using the QuikChange site-directed mutagenesis kit (Stra-

tagene) according to the manufacturer’s instructions with the primer

combination PIPK3 K442A QC for/PIPK3 K442A QC rev. The QuikChange

product was cloned into pGEM-Teasy (Promega), yielding PIPK3-K442A-

pGEM-Teasy. The cDNA encoding TripleA was amplified from PIPK3-

pGEM-Teasy using the primer combination PIPK3_loop_for/PIPK3-

loop4_rev and cloned into pGEM-Teasy, yielding the plasmid PIPK3-

TripleA-pGEM-Teasy. To create the DNT-MORN construct, the PIP5K3

cDNA sequence omitting the cDNA encoding the N terminus of PIP5K3 was

amplified using the primer combination AtPIP5K3Nco3_for/AtPIP5K3_

rev2, and the PCR product was cloned into the vector pGEM-Teasy,

yielding the plasmid DNT-MORN-PIP5K3-pGEM-Teasy. The cDNA en-

coding the truncated pip5k3-4-protein was amplified from PIPK3-pGEM-

Teasy using the primer combination AtPIPK3_Nco_for/PIPK3_as4.

PI4P 5-Kinase Promoter Fragments

For generation of promoter-GUS fusions, the GUSPlus gene was amplified

from the vector pCAMBIA1305.1 (accession number AF354045) using the

primer combination GUS_for/GUS_rev, and the PCR product was intro-

duced as a NotI-SacI fragment into the vector pgreen0029 (http://www.

pgreen.ac.uk/) (Hellens et al., 2000), yielding the plasmid pgreenGUSPlus.

Amplification of 1500-bp genomic sequences upstream of coding se-

quences foruse aspromoterswasachievedwithdifferent ArabidopsisBAC

clone templates as indicated, using the following primer combinations:

PromPIP5K1, PromPIP5K1_for/PromPIP5K1_rev from BAC clone F2E2;

PromPIP5K2, PromPIP5K2_for/PromPIP5K2_rev from BAC clone T32E8;

PromPIP5K3, PromPIP5K3_for/PromPIP5K3_rev from BAC clone T9J22;

PromPIP5K7, PromPIP5K7_for/PromPIP5K7_rev from BACclone T19D16;

PromPIP5K8, PromPIP5K8_for/PromPIP5K8_rev from BAC clone T7P1;

and PromPIP5K9, PromPIP5K9_for/PromPIP5K9_rev from BAC clone

F8A24. The PCR products representing PromPIP5K1, PromPIP5K3,

PromPIP5K7, PromPIP5K8, and PromPIP5K9 were moved directionally

as SalI-NotI fragments into the vector pgreenGUSPlus. The PCR pro-

duct representing PromPIP5K2 was moved as a NotI-NotI fragment into

pgreenGUSPlus. The resulting plasmids were transformed into Agro-

bacterium tumefaciens strain EHA105 and used for stable Arabidopsis

transformation.

PIP5K3 Overexpression Constructs

To create PIP5K3 overexpression constructs, an 800-bp fragment of the

characterized promoter sequence of the EXP7 gene (At1g12560) (Cho

and Cosgrove, 2002) was amplified using the primer combination

PromAtEXP7_for/PromAtEXP7_rev with genomic Arabidopsis DNA as a

template. The PCR product was moved as a SalI-NotI fragment into the

vector pUC18-Entry (Hornung et al., 2005), yielding the plasmid prom-

EXP7-puc18entry. The complete cDNA sequence of PIP5K3 was ampli-

fied with the primer combination PIP5K3Nco_for/PIP5K3_rev2 using the

plasmid PIP5K3-pGEM-Teasy as a template, and the PCR product was

cloned into pGEM-Teasy. The resulting plasmid was named PIP5K3-

pGEM-Teasy2. The PIP5K3-cDNA was moved as a NotI-NotI fragment

into the vector promEXP7-puc18entry, yielding the plasmid promEXP7-

PIP5K3puc18entry. To distinguish the overexpressed PIP5K3 clone from

the endogenous PIP5K3-allele, the cDNA sequence encoding EYFP was

amplified from the plasmid pEYFP (Clontech) using the primer combination

136 The Plant Cell

EYFP-Nco_for/EYFP-Nco_rev2 and moved as an NcoI-NcoI fragment in

frame with the PIP5K3 cDNA into the plasmid promEXP7-PIP5K3-

puc18entry, yielding promEXP7-YFP-PIP5K3puc18entry. The RedStar

(Janke et al., 2004) coding sequence was amplified from plasmids

carrying the authentic clones obtained from Martin Fulda using the primer

combination Redstar_for/Redstar_rev, cloned into pGEM-Teasy, and

moved from there as a NotI-NotI fragment first into the vector prom-

EXP7-puc18entry, yielding promEXP7-Redstar-puc18entry. For overex-

pression constructs for K442A and DNT-MORN, the respective clones

were moved as NotI-NotI fragments from the corresponding pGEM-Teasy

plasmids to promEXP7-puc18entry, yielding the plasmids promEXP7-

K442A-puc18entry and promEXP7-DNT-MORN-puc18entry, respectively.

Finally, the RedStar sequence without a stop codon was amplified using

the primer combination RedStar _for/RedStar_rev2 and moved directly as

an NcoI-NcoI fragment into promEXP7-K442A-puc18entry or promEXP7-

DNT-MORN-puc18entry, respectively. All entry clones were moved into the

vector pCambia 3300 using Gateway technology (Invitrogen) according to

the manufacturer’s instructions and transformed into the A. tumefaciens

strain EHA105.

Mutant Complementation Constructs

To create constructs for mutant complementation tests, the 1500-bp

promoter fragment promPIP5K3 was moved as a SalI-NotI fragment into

the vector pUC18-Entry (Hornung et al., 2005), yielding the plasmid

promPIP5K3puc18entry. The full-length PIP5K3 cDNA sequence was

moved from PIP5K3-pGEM-Teasy2 as a NotI-NotI fragment into the

vector promPIP5K3puc18entry, yielding the plasmid promPIP5K3-

PIP5K3puc18entry. To distinguish the introduced PIP5K3 clone from

the endogenous PIP5K3 allele, the cDNA sequence encoding EYFP

was moved as an NcoI-NcoI fragment in frame with the PIP5K3 cDNA

into the plasmid promPIP5K3-PIP5K3puc18entry, yielding the plas-

mid promPIP5K3-YFP-PIP5K3-puc18entry. The DNT-MORN, K442A, or

TripleA cDNA clones were moved as NotI-NotI fragments from their

respective pGEM-Teasy plasmids into theplasmidpromPIP5K3puc18entry,

yielding the plasmids promPIP5K3-DNT-MORN-puc18entry, promPIP5K3-

K442A-puc18entry, or promPIP5K3-TripleA-puc18entry, respectively, fol-

lowed by introduction of the EYFP sequence into the NcoI site of

these plasmids to produce promPIP5K3-YFP-DNT-MORN-puc18entry,

promPIP5K3-YFP-K442A-puc18entry, and promPIP5K3-YFP-TripleA-

puc18entry. All entry clones were moved into the vector pCambia 3300

using Gateway technology (Invitrogen) according to manufacturer’s instruc-

tions and transformed into the A. tumefaciens strain EHA105.

Bacterial Expression Constructs

Constructs for heterologous expression in Escherichia coli were created

by moving the cDNA sequences encoding PIP5K1, 3, 7, 8, and 9 as NcoI-NcoI

fragments from the plasmids PIP5K1-pGEM-Teasy, PIP5K3-pGEM-Teasy,

PIP5K7-pGEM-Teasy, PIP5K8-pGEM-Teasy, and PIP5K9-pGEM-Teasy,

respectively, into the bacterial expression vector pETM-41 (EMBL Protein

Expression and Purification Facility). The cDNA sequence of PIP5K2 was

released from the plasmid PIP5K2-pGEM-Teasy as a PciI-PciI fragment

and moved into the NcoI site of pETM-41. The DNT-MORN cDNA was

moved as an NcoI-NotI fragment from the vector DNT-MORN-PIP5K3-

pGEM-Teasy into the plasmid pETM-41. The cDNA encoding the

N-terminal MORN domain (PIP5K349-226) was amplified from PIP5K3-

pGEM-Teasy using the primer combination 59-GATCCATGGAGAAGG-

TGCTAAAGAACGGC-39/59-GATCCATGGCTCTTCCTCCCCCACCC-39.

The PCR product was cloned into pGEM-Teasy and moved from that

plasmid into pETM-41 as an NcoI-NcoI fragment. The coding sequences

for K442A, TripleA, and pip5k3-4 were moved as NcoI-NotI fragments

from the respective pGEM-Teasy plasmids into pETM-41. All clones were

introduced into the pETM-41 vector in frame with the cDNA encoding the

N-terminal MBP and polyhistidine (His) tags of pETM-41.

Fusion Constructs for Particle Bombardment

The cDNA clones encoding EYFP-PIP5K3, EYFP-K442A, and EYFP-

DNT-MORN were moved as NotI-NotI fragments into the vector

pENTR2b (Invitrogen). The coding sequence for the human PLCd1-PH

domain (Varnai and Balla, 1998) was amplified from plasmid DNA pro-

vided by Tamas Balla (National Institute for Child Health and Human

Development, Rockville, MD) and modified to encode a seven–amino

acid linker (Gly-Gly-Ala-Gly-Ala-Ala-Gly) between the PH domain and the

RedStar tag, as previously described (Dowd et al., 2006), using the primer

combination 59-GATCGCGGCCGCCGGTGGAGCTGGAGCTGCAGGA-

ATGAGGATCTACAGGCGC-39/59-GATCGATATCTTAGATCTTGTGCAG-

CCCCAGCA-39. The amplicon was moved into pENTR2b as a NotI-

EcoRV fragment. The pENTR2b constructs were transferred by Gateway

technology (Invitrogen) from pENTR2b to the plasmid pLatGW, an ex-

pression vector containing the tomato Lat52 promoter (Twell et al., 1990),

an attR Gateway cassette, and a pA35S terminator. The pLatGW-vector

was a gift from Wolfgang Droge-Laser (University of Gottingen, Germany).

Detection of Specific Transcripts and Analysis of PIP5K3 Expression

Levels by RT-PCR

Expression levels of PIP5K3 in wild-type plants and in pip5k3-1, pip5k3-2,

pip5k3-3, and pip5k3-4 mutants were analyzed by RT-PCR. Total RNA

was extracted from roots, stems, leaves, and flowers using Plant RNA

purification reagent (Invitrogen). For removal of contaminating genomic

DNA, RNA samples were incubated for 30 min at 378C with RNase-free

DNaseI, and the RNA was subsequently precipitated. Five micrograms of

total RNA were reverse transcribed with RevertAid H Minus M-MuLV

reverse transcriptase (Fermentas) in the presence of oligo(dT) primers.

For determination of relative transcript levels, equal amounts of first-

strand cDNA were used as templates for PCR amplification using the

primer combination PIP5K3Nco_for/PIP5K3Nco_rev. The Arabidopsis

actin8 (ACT8) gene was amplified using the primer combination

AtACT8_F/AtACT8_R and served as an internal positive control (Bustin,

2000). Truncated transcripts as shown in Figure 5H were detected using

the primer combination YFP_s/PIPK3Nco_rev. Transcript levels shown in

Figure 7B were detected using the following primer combinations: Red-

Star, RedStar_for/RedStar_rev; K442A and DNT-MORN, RedStar-s/

PIP5K3Nco_rev.

Determination of Specific Transcript Levels by Quantitative

Real-Time RT-PCR

The levels of specific transcripts in roots were determined by real-time

RT-PCR analysis of cDNA reverse transcribed from 1 mg of total RNA

using 100 units of Reverse Transcriptase H� (MBI Fermentas) according

to the manufacturer’s instructions. The resulting cDNA was diluted 1:10,

and 1 mL was used as a PCR template in a 25-mL reaction containing 2.5

mL 103 PCR buffer (Bioline), 2 mM MgCl2, 100 mM deoxynucleotide

triphosphate, 2.5 mL QuantiTect primer mix (Qiagen), 0.1-fold Sybr green,

10 nM fluorescein, and 0.25 units of BioTaq (Bioline). Samples were

denatured for 3 min at 958C, followed by 40 cycles of 20-s denaturation at

958C, 20 s of annealing at 558C, and 40 s of elongation at 728C.

Fluorescence was monitored during each annealing and denaturation

phase. The program was concluded by 4 min of elongation at 728C and

1 min of denaturation at 958C. Renaturing of amplified DNA was followed

by the assessment of melting parameters by increasing the temperature

in 0.58C increments while monitoring fluorescence. In addition to the

individual QuantiTect primers used, a DNA fragment of the PP2A subunit

PI4P 5-Kinase 3 and Root Hair Development 137

of PDF2 (At1g13320) was used as an internal reference. Transcript levels

were calculated according to Livak and Schmittgen (2001).

Heterologous Expression in E. coli

Recombinant enzymes were expressed in E. coli strain BL21-AI (Invitro-

gen) at 258C for 18 h after induction with 1 mM isopropylthio-b-galacto-

side and 0.2% (w/v) L-arabinose. The PIP5K3-MORN domain was

expressed in E. coli Rosetta II cells (Novagen) at 258C for 4 h after

induction with 1 mM isopropylthio-b-galactoside. Cell lysates were

obtained by sonification in a lysis buffer containing 50 mM Tris-HCl,

300 mM NaCl, 1 mM EDTA, and 10% (v/v) glycerol, pH 8.0.

Lipid Kinase Assays

Lipid kinase activity was assayed by monitoring the incorporation of

radiolabel from [g-32P]ATP into defined lipid substrates according to Cho

and Boss (1995) using total extracts of BL-21-AI expression cultures.

Recombinant expression levels were adjusted between individual cul-

tures according to immunodetection of expressed MBP-tagged proteins

(data not shown), allowing for the comparison of enzyme activities

obtained with different cultures. Radiolabeled lipid reaction products

were separated by thin layer chromatography and visualized by autora-

diography using Kodak X-Omat autoradiography film (Eastman Kodak).

Reaction products were identified according to comigration with authen-

tic standards (Avanti Polar Lipids). Phosphatidylinositol-bisphosphate

bands were scraped according to autoradiography, and incorporated

radiolabel was quantified using liquid scintillation counting (Analyzer

Tricarb 1900 TR; Canberra Packard). Enzyme activites presented in

Figure 2, Table 1, and Supplemental Figure 2 online represent the mean of

at least three independent experiments, assayed in duplicates.

Arabidopsis Transformation

Recombinant constructs were introduced into Arabidopsis plants through

A. tumefaciens–mediated transformation using the floral dip method

(Clough and Bent, 1998). Independent transformants were subjected to

selective conditions on MS medium containing either 50 mg mL�1

kanamycin (for promoter-GUS plants) or 10 mg mL�1 glufosinate-ammo-

nium (for complementation constructs in T-DNA insertion lines), 1% (w/v)

sucrose, and 0.7% (w/v) agar. Resistant seedlings were transferred to soil

after 2 to 3 weeks; homozygous T2 plants were used for analysis.

Histochemical Staining for GUS Activity

Histochemical staining of plant tissue for GUS activity was performed as

previously described (Jefferson et al., 1987). In brief, tissue samples were

vacuum-infiltrated for 5 min in a GUS substrate solution of 100 mM

sodium phosphate, pH 7.0, 2 mM 5-bromo-4-chloro-3-indolyl-glucu-

ronide, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide,

10 mM EDTA, and 0.1% (v/v) Triton X-100 and then incubated at 378C for

3 h. Subsequently, the samples were transferred to 70% ethanol to

remove chlorophyll pigmentation. GUS-positive samples were examined

with a bright-field microscope (Olympus BX51) or a stereomicroscope

(Olympus SZX12) at low magnification (34 to 310), and digital images

were recorded. For cross sections, stained roots were embedded in

polyethylene glycol 1500 according to Hause et al. (1996). Cross sections

of 10 mm thickness were cut with a microtome (HM 355; Microm

International), transferred to poly-L-Lys–coated slides, and examined by

bright-field microscopy using an AxioImager microscope (Zeiss). Micro-

graphs taken with AxioCam MRc5 (Zeiss) were processed through

Photoshop 8.0.1 (Adobe Systems). All GUS-stained samples shown

represent typical results of at least three independent transgenic lines for

each construct.

Quantification of Root Hair Length

Root hair length from 8-d-old plants grown on agar plates was deter-

mined on low-magnification (310) digital images captured using a CCD

camera (ColorViewII) and image analysis freeware (ImageJ; http://rsb.

info.nih.gov/ij/). To ensure comparable results, the area 3 to 5 mm behind

the root tip was analyzed. Plants grown on agar plates were carefully

removed in ;100 mL of half-strength MS medium (Duchefa) on micro-

scope slides for analysis. Quantification data are the means of 200 to 350

values representing 10 root hairs each of 20 to 35 individual plants

measured for each data point.

Pollen Tube Growth and Transient Gene Expression

Mature pollen was collected from four to six tobacco (Nicotiana tabacum)

flowers of 8-week-old plants. Pollen was resuspended in growth medium

(Read et al., 1993), filtered onto cellulose acetate filters, and transferred to

Whatman paper moistened with growth medium. Within 5 to 10 min of

harvesting, pollen was transformed by bombardment with plasmid-

coated 1-mm gold particles with a helium-driven particle accelerator

(PDS-1000/He; Bio-Rad) using 1350-psi rupture discs and a vacuum of

28 inches of mercury. Gold particles (1.25 mg) were coated with 3 to 7 mg

of plasmid DNA. After bombardment, pollen was resuspended in growth

medium and grown for 7 to 10 h in small droplets of media directly on

microscope slides.

Microscopy and Imaging

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 (MBS) and a 530 to 600-nm band-pass filter; RedStar

was excited at 561 nm and imaged using an HFT 405/488/561 nm MBS

and a 583 to 604-nm band-pass filter; EYFP and FM4-64 were synchro-

nously excited at 488 nm and 561 nm, respectively, and imaged using an

HFT 405/488/561-nm MBS and a 518 to 550-nm band-pass filter and a

657 to 754-nm band-pass filter, respectively. Root hair autofluorescence

was imaged using an HFT 405/514/633-nm MBS and a 470 to 500-nm

band-pass filter to record autofluorescence without fluorescence cross-

leakage from the EYFP channel. The autofluorescence signal was then

subtracted from the EYFP signal. Fluorescence and transmitted light

images were contrast-enhanced by adjusting brightness and g-settings

using image-processing software (Photoshop; Adobe Systems), except

where stated differently. Fluorescence intensities were determined using

AnalySIS software (Olympus). FM4-64 was added to pollen tubes at a

final concentration of 10 mM as described (Parton et al., 2001) and

visualized after 5 to 15 min of incubation before dye internalization.

Accession Numbers

Arabidopsis Genome Initiative locus identifiers of genes used in

this study are as follows: PIP5K1, At1g21980; PIP5K2, At1g77740;

PI5K3, At2g26420; PIP5K7, At1g10900; PIP5K8, At1g60890; PIP5K9,

At3g09920; ACT8, At1g49240; PDF2 (PP2A), At1g13320.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Organ-Specific Expression Patterns of PI4P

5-Kinase Isoforms 2 and 9 in Arabidopsis.

Supplemental Figure 2. Catalytic Activity of Arabidopsis PI4P

5-Kinase Isoforms Expressed in Roots.

Supplemental Figure 3. Identification of Homozygous T-DNA Inser-

tion Mutants in the PIP5K3 Gene Locus At2g26420.

138 The Plant Cell

Supplemental Figure 4. Subcellular Localization of PIP5K3 and

K442A Heterologously Expressed in Tobacco Pollen Tubes.

Supplemental Table 1. Primers Used in This Study.

Supplemental Text and References.

ACKNOWLEDGMENTS

We thank the following individuals: Tamas Balla (National Institute for Child

Health and Human Development, Rockville, MD) for the HsPLCd1-PH

domain construct, Andreas Hiltbrunner (Eidgenossische Technische

Hochschule, Zurich, Switzerland) for the pCambia vector, Wolfgang

Droge-Laser and Martin Fulda for cDNA constructs, Susanne Mesters

for expert plant culture, Andreas Wodarz and Michael Krahn for access

to the confocal microscope and technical support, respectively, Linh Vu

Hai for technical assistance (all University of Gottingen, Germany). We

also thank Imara Perera, Wendy Boss, and Yang Ju Im (all North

Carolina State University, Raleigh, NC) and Ivo Feussner (University of

Gottingen, Germany) for helpful discussion and critical reading of

this manuscript. We gratefully acknowledge financial support by the

ERASMUS/SOCRATES program (to A.H. and M.S.) and an Emmy

Noether grant from the German Research Foundation (to I.H.).

Received May 15, 2007; revised December 13, 2007; accepted Decem-

ber 18, 2007; published January 4, 2008.

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PI4P 5-Kinase 3 and Root Hair Development 141

DOI 10.1105/tpc.107.052852; originally published online January 4, 2008; 2008;20;124-141Plant Cell

HeilmannIrene Stenzel, Till Ischebeck, Sabine König, Anna Holubowska, Marta Sporysz, Bettina Hause and Ingo

Arabidopsis thalianaThe Type B Phosphatidylinositol-4-Phosphate 5-Kinase 3 Is Essential for Root Hair Formation in

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