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. 2008 Feb;20(2):381–395. doi: 10.1105/tpc.107.054304

ROOT HAIR DEFECTIVE4 Encodes a Phosphatidylinositol-4-Phosphate Phosphatase Required for Proper Root Hair Development in Arabidopsis thaliana[W]

Julie M Thole a, Joop EM Vermeer b, Yanling Zhang c, Theodorus WJ Gadella Jr d, Erik Nielsen a,c,1
PMCID: PMC2276440  PMID: 18281508

Abstract

Polarized expansion of root hair cells in Arabidopsis thaliana is improperly controlled in root hair–defective rhd4-1 mutant plants, resulting in root hairs that are shorter and randomly form bulges along their length. Using time-lapse fluorescence microscopy in rhd4-1 root hairs, we analyzed membrane dynamics after labeling with RabA4b, a marker for polarized membrane trafficking in root hairs. This revealed stochastic loss and recovery of the RabA4b compartment in the tips of growing root hairs, consistent with a role for the RHD4 protein in regulation of polarized membrane trafficking in these cells. The wild-type RHD4 gene was identified by map-based cloning and was found to encode a Sac1p-like phosphoinositide phosphatase. RHD4 displayed a preference for phosphatidylinositol-4-phosphate [PI(4)P] in vitro, and rhd4-1 roots accumulated higher levels of PI(4)P in vivo. In wild-type root hairs, PI(4)P accumulated primarily in a tip-localized plasma membrane domain, but in rhd4-1 mutants, significant levels of PI(4)P were detected associated with internal membranes. A fluorescent RHD4 fusion protein localized to membranes at the tips of growing root hairs. We propose that RHD4 is selectively recruited to RabA4b-labeled membranes that are involved in polarized expansion of root hair cells and that, in conjunction with the phosphoinositide kinase PI-4Kβ1, RHD4 regulates the accumulation of PI(4)P on membrane compartments at the tips of growing root hairs.

INTRODUCTION

The establishment of polarity is an essential aspect of eukaryotic cellular differentiation and requires asymmetric delivery of membranes and proteins via membrane trafficking. While much is known about the organization of individual membrane trafficking pathways within cells, less is known about how they are regulated during cellular differentiation. We have focused specifically on the process of polarized membrane trafficking during the differentiation of epidermal root hair cells in Arabidopsis thaliana.

Arabidopsis root hairs are single cells that grow by targeted secretion of cell wall components to the growing tip of the cell (Schnepf, 1986; Dolan, 2001). In plants, polarized cell growth requires new cell wall deposition and is dependent upon polarized secretion of vesicle cargo, with Rab GTPases mediating the sorting and delivery of this cargo within the cellular membrane trafficking pathway. Rab GTPases function by recruiting cytosolic effector proteins to specific subcellular membranes to regulate aspects of membrane trafficking through those compartments (Zerial and McBride, 2001).

We have previously shown that RabA4b, a plant Rab GTPase, is localized to membrane compartments at the tips of growing root hairs, where it functions to regulate polarized secretion (Preuss et al., 2004). We also recently reported that phosphatidylinositol-4 kinase β1 (PI-4Kβ1), a RabA4b effector protein, is necessary for proper root hair development (Preuss et al., 2006). Therefore, RabA4b and its effector PI-4Kβ1 are required for proper polarized secretion in root hairs in Arabidopsis. Similarly, interaction of Rab GTPases and phosphoinositide kinases has been shown in other systems: mammalian Rab11 interacts directly with PI4KIIIβ (de Graaf et al., 2004), and yeast Ypt31 interacts genetically with Pik1p (Sciorra et al., 2005).

In eukaryotic cells, particular phosphoinositides preferentially mark subcellular membranes and recruit phosphoinositide binding proteins, providing spatial and temporal cues within the cell (Bankaitis and Morris, 2003; Pendaries et al., 2005). In both yeast and animal cells, generation and turnover of phosphoinositide pools play important roles in regulation of membrane trafficking steps (De Matteis and Godi, 2004; Di Paolo and De Camilli, 2006). In particular, in animals, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] play essential roles in cellular polarization in a number of differentiating cell types (Pinal et al., 2006; Martin-Belmonte et al., 2007; Nishio et al., 2007). In plants, PI(4,5)P2 accumulates at the tips of growing pollen tubes and root hair cells (Kost et al., 1999; Vincent et al., 2005), and mutation of a phosphatidylinositol transfer protein results in shorter, branched root hairs (Bohme et al., 2004; Vincent et al., 2005). Also, phospholipase C (PLC) uses PI(4,5)P2 as a substrate, and mutations in PLC influence pollen tube growth and root hair growth (Fischer et al., 2004; Dowd et al., 2006).

Yeast Sac1p is an essential gene in yeast, and this gene has been characterized as a phosphatidylinositol-4-phosphate (PI(4)P) phosphatase (Hughes et al., 2000; Foti et al., 2001). The SAC phosphatase domain has been defined by the seven highly conserved motifs found in Sac1p (Hughes et al., 2000). Yeast also contains a second SAC domain–containing phosphatase, Fig4p, which is a PI(3,5)P2 5-phosphatase (Rudge et al., 2004). SAC domains are also found in mammalian synaptojanins and the yeast homologs Inp52p and Inp53p. These proteins contain a 5-phosphatase domain in addition to their N-terminal SAC domain (Hughes et al., 2000).

The Arabidopsis genome contains nine SAC domain proteins, which can be separated into three classes: SAC1-5 are most similar to yeast Fig4p, SAC6-8 are most similar to yeast Sac1p, and SAC9 contains unique domains (Zhong and Ye, 2003). Thus far, only two of these have been characterized: The SAC1 protein has been shown to have PI(3,5)P2 phosphatase activity, and mutation of the SAC1 gene causes cell wall and actin cytoskeleton defects along with aberrant cell morphology (Zhong et al., 2005). Mutation of SAC9 results in overaccumulation of PI(4,5)P2 and induces expression of stress response genes (Williams et al., 2005). Less is known about the SAC6-8 genes except that their cDNAs can rescue the yeast Sac1p mutation (Despres et al., 2003). Additionally, both a promoter-β-glucuronidase study and tissue-specific RT-PCR experiments (Despres et al., 2003; Zhong and Ye, 2003) demonstrate that SAC7 and SAC8 are expressed broadly, while SAC6 is expressed only in pollen.

Previously, we found an altered localization of enhanced yellow fluorescent protein (EYFP)-RabA4b fusion protein in short bulged root hairs of the mutant rhd4-1 (Schiefelbein and Somerville, 1990); EYFP-RabA4b was not observed in bulging root hairs in rhd4-1 but appeared to be tip localized in root hairs with tip-restricted expansion or in those in which tip growth had reestablished from bulges (Preuss et al., 2004). Here, we use time-lapse fluorescence microscopy to show that tip localization of EYFP-RabA4b–labeled membrane compartments display altered dynamics in rhd4-1 root hairs. Using map-based cloning techniques, we identified RHD4 as a Sac1p-like phosphoinositide phosphatase, previously named SAC7. In vitro experiments demonstrate that RHD4 displays selectivity for PI(4)P, and rhd4-1 mutant plants contain increased levels of PI(4)P in root tissues. Correspondingly, by monitoring localization of a PI(4)P binding RHD fusion protein, we observed an altered localization of PI(4)P in rhd4-1 root hairs. Based on these observations and the previous identification of a role for PI-4Kβ1 in tip growth, we propose that regulation of both PI(4)P levels and its subcellular distribution by RHD are important for proper organization of polarized secretion during root hair development.

RESULTS

Spatial and Temporal Distribution of RabA4b Is Altered in rhd4-1 Root Hairs

Previously, we showed that the plant Rab GTPase, RabA4b, labeled membranes that accumulated at the tips of growing root hair cells in Arabidopsis (Preuss et al., 2004). To better understand the role of RabA4b in root hair development, we expressed a fusion of EYFP and RabA4b in a series of root hair mutant backgrounds (Preuss et al., 2004).

To determine if tip localization of EYFP-RabA4b–labeled membranes was tightly linked with reestablished tip-restricted expansion out of root hair bulges, we performed time-lapse fluorescence microscopy in both wild-type and rhd4-1 root hairs. In wild-type root hairs, the localization and levels of EYFP-RabA4b membranes detected by fluorescence microscopy in the tips of growing root hairs remained consistent over time (Figures 1A and 1B; see Supplemental Movie 1 online). By contrast, the presence and levels of EYFP-RabA4b membranes detected in the tips of rhd4-1 root hairs varied greatly over time (Figure 1C; see Supplemental Movie 2 online). In many cases, loss of tip-localized EYFP-RabA4b compartments correlated with bulge formation and impaired rates of elongation (Figures 1C and 1D, arrows). When tip growth resumed from these bulges, it was accompanied by the accumulation of tip-localized EYFP-RabA4b (Figure 1C and shaded region in Figure 1D).

Figure 1.

Figure 1.

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Spatial and Temporal Distribution of RabA4b Is Altered in rhd4-1 Mutants.

Wild-type and rhd4-1 Arabidopsis seedlings expressing EYFP-RabA4b were visualized by time-lapse fluorescence microscopy, and frames from a 20-min period are shown, with a reference line for root hair length at time 0. Relative length was measured from time 0 as 0 μm. The fluorescent signal located within the proximal 7% of the length of the root hair was defined as tip fluorescence, and this was presented as a percentage of the fluorescence detected within the entire root hair.

(A) EYFP-RabA4b was localized to the tip of a growing wild-type root hair over a time period of 20 min.

(B) Quantification of EYFP-RabA4b localization in a wild-type root hair. Black line, relative length of the root hair in micrometers; gray line, percentage of tip localization of the fluorescent signal.

(C) EYFP-RabA4b localization is altered in a growing rhd4-1 root hair. Arrows indicate time points at which the root hair is bulging, and EYFP-A4b localization is lost.

(D) Quantification of EYFP-RabA4b localization in an rhd4-1 root hair. Root hair tip growth correlates with increased EYFP-RabA4b tip fluorescence (shaded regions). Arrows indicate time points at which the root hair is bulging, and EYFP-A4b localization is lost. Black line, relative length of the root hair in micrometers; gray line, percentage of tip localization of the fluorescent signal.

When quantifying this type of growth, we observed a relationship between the rate of root hair elongation and the presence of florescence signal obtained from the EYFP-RabA4b compartment (Figure 1D). We have observed this type of altered localization in >20 root hairs, and we have shown the best example of this localization pattern. While it was not clear whether loss or recovery of the tip-localized RabA4b compartment was responsible for bulge formation, this stochastic loss and recovery of the tip-localized compartment was clearly associated with the halting and resumption of elongation of the root hair and the bulged phenotype. These results suggested that RHD4 may be involved in regulating either proper distribution and/or accumulation of tip-localized RabA4b compartments during polarized growth in these cells.

RHD4 Is a Sac1p-Like Phosphoinositide Phosphatase

Based on the spatial and temporal localization pattern of EYFP-RabA4b in rhd4-1 root hairs, we hypothesized that RHD4 may play a role in coordinating delivery of cell wall materials to the tips of root hairs. To determine RHD4 function, we undertook a map-based cloning approach to identify the gene responsible for the rhd4-1 mutation, which had previously been mapped to the lower arm of chromosome 3 (Wang et al., 2001). To determine the genetic location of the RHD4 locus, a mapping population was generated by crossing rhd4-1 with the Arabidopsis ecotype Landsberg erecta. Homozygous rhd4-1 F2 seedlings were selected, and simple sequence length polymorphisms (SSLPs) and cleaved amplified polymorphic sequences (CAPS) were used for physical mapping of the rhd4-1 locus to an ∼100 kb region of chromosome 3 containing 60 predicted open reading frames (Figure 2A).

Figure 2.

Figure 2.

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RHD4 Encodes a Sac1p-Like Phosphoinositide Phosphatase.

(A) Map-based cloning of the rhd4 mutation. RHD4 is located on the right arm of chromosome 3, At3g51460. For spatial reference, described genetic markers are shown with their corresponding map position in centimorgans (cM). The rhd4-1 allele had a G→A base pair substitution at the first base of the first intron. The rhd4-2 allele had a mutation in exon 16 causing an amino acid substitution of Gly to Ser, G449S, in the protein. Two SALK T-DNA insertional lines were obtained, designated rhd4-3 and rhd4-4, and these had insertions in the second and fourth introns, respectively.

(B) Amino acid alignment of the SAC domains of RHD4 and other SAC domain proteins from yeast and animals. The SAC domain sequences of yeast Sac1 (Sac1p), human Sac2 (hSAC2), rat SAC1 (rSAC1), human synaptojanin (Synapto), and RHD4 were aligned using ClustalW. Identical amino acids are shaded black, and similar amino acids are shaded gray. The seven conserved motifs of the SAC domain (Hughes et al., 2000) are marked by solid lines above the sequences. The location of the rhd4-2 G449S amino acid substitution in motif VII is marked by an asterisk.

Several candidate genes in this region were sequenced, including At3g51460, which was predicted to encode a Sac1p-like phosphoinositide phosphatase protein. The SAC domain of this protein, previously designated At SAC7 (Zhong and Ye, 2003), contains all seven of the conserved motifs of yeast Sac1p (Figure 2B). In sequencing the rhd4-1 allele, a G→A base pair substitution was identified in At3g51460 at the first base of the first intron, which would prevent proper intron splicing. A second ethyl methanesulfonate (EMS)–generated rhd4 allele, designated rhd4-2, contained a G449S amino acid substitution in motif VII of the SAC phosphatase domain (asterisk in Figure 2B). Similar mutations in this conserved region were previously isolated and shown to disrupt phosphatase activity in yeast Sac1p (Hughes et al., 2000). Therefore, both the rhd4-1 and rhd4-2 alleles contained mutations that would potentially inactivate the phosphoinositide phosphatase protein encoded by At3g51460.

To confirm the identification of RHD4 as At3g51460, SALK T-DNA insertion lines were obtained (Alonso et al., 2003) and analyzed for mutant root hair phenotypes. Two homozygous lines were identified and were designated rhd4-3 and rhd4-4, which have insertions located in the second and fourth introns, respectively (Figure 2A). RNA was isolated from seedlings of the wild type and all four rhd4 alleles, and cDNA was amplified for RT-PCR. Transcript levels were significantly reduced in rhd4-1, rhd4-3, and rhd4-4, although low levels of transcript could be detected with increased PCR cycles (Figure 3A). As expected, rhd4-2 transcript levels are similar to the wild type, as this allele simply contains a base pair substitution.

Figure 3.

Figure 3.

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RHD4 Alleles and Complementation.

(A) RT-PCR of RHD4 expression in wild-type seedlings and in mutant seedlings containing each of the different rhd4 alleles. RNA was extracted from seedlings for cDNA synthesis and RT-PCR. In the rhd4-1 sample, a larger band was observed (asterisk), and this band corresponds to the RHD4 transcript including the first intron due to the G→A base pair substitution, which prevents proper splicing. β-tubulin is used as an internal loading control.

(B) Phenotypes caused by the rhd4 mutant alleles. Bars = 100 μm.

(C) Complementation of the rhd4-1 mutation. rhd4-1 mutant Arabidopsis plants were transformed by Agrobacterium-mediated transformation with an At3g51460 native promoter-At3g51460 cDNA construct (left panel) and a native promoter-EYFP-cDNA construct observed by light microscopy (middle panel) and fluorescence microscopy (right panel). Bars = 100 μm.

(D) Quantification of root hair length. Root hairs of wild-type, rhd4 alleles, and complemented seedlings were measured (n = 314, 311, 272, 268, 308, 464, and 398 root hairs, respectively). Error bars represent 1 sd.

Since low levels of two differently sized transcripts were detected in the rhd4-1 mutant, sequences were obtained for both to determine if any wild-type transcript was present. Both low-level transcripts were nonfunctional, with the larger transcript (Figure 3A, asterisk) containing the entire first intron due to the rhd4-1 splicing mutation. The smaller transcript had an improperly spliced intron, and both transcripts resulted in premature stop codons (see Supplemental Figure 1 online). Therefore, all four of the rhd4 alleles we characterized are either nonfunctional (rhd4-1 and rhd4-2) or expression is virtually eliminated (rhd4-3 and rhd4-4). All four alleles have the same phenotype of short, bulged, and branched root hairs (Figure 3B). Quantification of root hair length demonstrated that all four alleles have significantly shorter root hairs (Figure 3D), and all four alleles have a similar percentage of morphologically aberrant root hairs (Table 1).

Table 1.

Root Hair Morphology of rhd4 Alleles and Complemented Lines

Wild Type Branched Bulged Popsicle Wavy Jagged
Wild type 94.27 ± 2.26 0.80 ± 0.81 1.88 ± 0.96 0.09 ± 0.26 0.29 ± 0.43 2.68 ± 1.95
rhd4-1 45.69 ± 8.56 5.35 ± 1.81 39.69 ± 8.11 0.85 ± 1.55 3.63 ± 3.55 4.79 ± 2.05
rhd4-2 55.59 ± 14.18 6.89 ± 4.27 30.55 ± 6.54 1.00 ± 1.10 1.04 ± 1.03 5.56 ± 6.17
rhd4-3 35.68 ± 14.00 8.49 ± 5.89 46.16 ± 6.31 0.00 1.34 ± 0.88 8.33 ± 3.72
rhd4-4 39.59 ± 8.89 7.01 ± 2.68 45.09 ± 10.48 0.75 ± 2.11 1.77 ± 1.15 5.79 ± 1.54
Complemented 85.07 ± 7.13 6.49 ± 2.71 4.08 ± 2.82 0.00 1.23 ± 1.53 3.12 ± 1.42
EYFP-complemented 90.89 ± 5.97 1.02 ± 0.73 1.45 ± 1.42 0.00 1.02 ± 0.73 5.62 ± 4.52
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Percentages of each root hair class (defined in Preuss et al., 2006) were determined for the wild type, all four rhd4 alleles, the native promoter-cDNA complemented line, and the native promoter-EYFP-cDNA complemented line (n = 1152, 1720, 1529, 1365, 1482, 983, and 947 root hairs, respectively). Errors represent the sd of the average for each line.

Finally, genetic complementation was used to demonstrate that At3g51460 was indeed the gene responsible for the rhd4-1 mutation. We introduced wild-type At3g51460 into the rhd4-1 background using Agrobacterium tumefaciens–mediated transformation. Expression of wild-type At3g51460 in the rhd4-1 mutant background under the control of its own promoter resulted in fully expanded, morphologically wild-type root hairs (Figure 3C). Expression of an EYFP-At3g51460 fusion protein from its own promoter also rescued the rhd4-1 mutant phenotype (Figure 3C). Quantification of root hair length demonstrated that both the cDNA-complemented line and the EYFP-cDNA–complemented line have significantly longer root hairs than the uncomplemented rhd4 alleles (Figure 3D), and both lines have root hairs with a restored wild-type morphology (Table 1).

RHD4 Is a PI(4)P Phosphatase

Based on sequence similarity, RHD4 was predicted to encode a phosphoinositide phosphatase. To test this, we expressed and purified both wild-type RHD4 and the mutant rhd4-2 N-terminal catalytic domains from Escherichia coli as glutathione S-transferase (GST) fusion proteins for an in vitro phosphatase activity assay. As RHD4 was most similar to the yeast Sac1p, which has been shown to have PI(4)P phosphatase activity (Hughes et al., 2000; Foti et al., 2001; Zhong and Ye, 2003), we first tested whether purified RHD4 had PI(4)P phosphatase activity. GST-RHD4 displayed higher levels of PI(4)P phosphatase activity than GST alone (Figure 4A). Additionally, the rhd4-2 allele containing the G449S mutation in the phosphatase domain reduced the PI(4)P phosphatase activity to the residual levels observed for GST alone (Figure 4A). To determine if in vitro PI phosphatase activity detected for GST-RHD4 was specific for PI(4)P, we also tested whether RHD4 displayed phosphatase activity for other PI isoforms. While we did observe some limited phosphatase activity with different phosphoinositides, RHD4 had significantly higher phosphatase activity against PI(4)P (Figure 4B), consistent with this protein being a PI(4)P phosphatase.

Figure 4.

Figure 4.

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RHD4 Is a PI(4)P Phosphatase.

(A) GST-RHD4 has high levels of PI(4)P phosphatase activity in vitro. GST, GST-RHD4, and GST-rhd4-2 were expressed and purified from E. coli. Error bars represent the sd of two biological replicates.

(B) GST-RHD4 has low levels of activity against all other phosphoinositide substrates tested. Values are calculated with PI(4)P activity as 100%. For each substrate, phosphatase release was measured for GST and GST-RHD4; the GST values were then subtracted from GST-RHD4 values to eliminate background. Error bars represent the sd of two replicates.

(C) rhd4-1 roots accumulate PI(4)P. Two-week-old wild-type and rhd4-1 seedlings were transferred to 0.25× Murashige and Skoog (MS) media supplemented with 3H-myoinositol for 40 h. Radiolabeled phospholipids were isolated from root tissue, deacylated, and fractionated with HPLC. PI(4)P levels were calculated as a percentage of total PI. Error bars represent the sd from the average of nine samples.

(D) Representative traces from the HPLC of 3H-labeled phospholipids from rhd4-1 and wild-type roots. The glycerophosphoinositol peaks (gPI) for gPI(3)P and gPI(4)P are shown. rhd4-1 (black line) has elevated levels of PI(4)P compared with the wild type (gray line), while PI(3)P levels are comparable between the wild type and rhd4-1.

While many PI phosphatases have been shown to demonstrate activity against a broad number of substrates in vitro, their in vivo activities may be quite different due to restricted access to substrates based on their subcellular localization (Hughes et al., 2000). Therefore, to confirm that RHD4 also had PI(4)P phosphatase activity in vivo, we assessed levels of phosphoinositides present in wild-type and rhd4-1 mutant roots. Wild-type and rhd4-1 seedlings were grown in the presence of 3H-inositol, and phospholipids were extracted from root tissues for comparison by HPLC. In rhd4-1 roots, PI(4)P levels were ∼50% higher than those in wild-type roots (Figures 4C and 4D). Based on these results, we concluded that RHD4 acts as a PI(4)P phosphatase in vivo and that loss of this activity in rhd4 mutants results in overaccumulation of this phosphoinositide isoform.

PI(4)P Localization Is Altered in rhd4-1 Root Hairs

Because RHD4 displayed PI(4)P phosphatase activity in vitro and in vivo, we were interested whether loss of this activity would result in altered accumulation or distribution of PI(4)P within developing root hairs. Therefore, we were interested in observing localization of PI(4)P pools in root hairs. We decided to create an EYFP biosensor that would bind to pools of PI(4)P in living plants. These types of PI binding biosensors have been widely used in mammalian systems (Varnai and Balla, 2007). We compared the in vivo binding of the well-characterized mammalian proteins FYVE and FAPP1. A tandem repeat of the FYVE domain (2XFYVE) has been used to visualize PI(3)P in mammalian cells (Gillooly et al., 2000) and in Arabidopsis root hairs, where PI(3)P has been localized to endosomal compartments (Voigt et al., 2005b). The PH domain of FAPP1 has been used to visualize PI(4)P in mammalian cells (Godi et al., 2004; Balla et al., 2005). In in vitro lipid binding assays, GST-2XFYVE showed specific binding to PI(3)P, and GST-FAPP1 showed specific binding to PI(4)P (Figure 5A). GST alone does not bind any of the lipids.

Figure 5.

Figure 5.

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EYFP-FAPP1 Is Localized to Subcellular Membrane Compartments in rhd4-1 Root Hairs.

(A) GST, GST-2XFYVE, and GST-FAPP1 lipid binding assays.

(B) GFP-FYVE, a PI(3)P binding protein, is localized to the endosomal compartment in wild-type root hairs. A z-stack projection is shown. Bar = 10 μm.

(C) GFP-FYVE localization in the rhd4-1 background. A z-stack projection is shown. Bar = 10 μm.

(D) EYFP-FAPP1 is localized to the tips of growing wild-type root hairs. Arrowheads show localization to some subcellular membrane compartments. A z-stack projection of a wild-type root hair is shown. Bar = 10 μm.

(E) EYFP-FAPP1 is enriched on the plasma membrane of wild-type root hairs. Cross sections were made through the z-stack projection at various positions in the tip region as represented by solid white lines. Cross sections were then used to quantify the amounts of EYFP-FAPP1 along a line, represented by the dashed white line.

(F) Quantification of EYFP-FAPP in wild-type cross sections. ImageJ was used to quantify the pixel intensity of EYFP-FAPP1 along a vertical line through the center of each cross section (dashed white line in [E]). Each pixel intensity plot corresponds to the cross section above it in (E).

(G) EYFP-FAPP1 is accumulated on subcellular membrane compartments in rhd4-1 root hairs. A z-stack projection of a wild-type root hair is shown. Bar = 10 μm.

(H) EYFP-FAPP1 is found on subcellular membrane compartments throughout rhd4-1 root hairs (arrowheads). Cross sections were made through the z-stack projection at various positions in the tip region as represented by solid white lines. Cross sections were then used to quantify the amounts of EYFP-FAPP1 along a line, represented by the dashed white line.

(I) Quantification of EYFP-FAPP1 in rhd4-1 cross sections. ImageJ was used to quantify the pixel intensity of EYFP-FAPP1 along a vertical line through the center of each cross section (dashed white line in [H]). Each pixel intensity plot corresponds to the cross section above it in (H).

GFP-2XFYVE was localized to the endosomal compartment throughout wild-type root hairs (Figure 5B), and its distribution was unchanged in the rhd4-1 background (Figure 5C), demonstrating that PI(3)P localization is not altered by mutation of the RHD4 phosphatase. In wild-type root hairs, EYFP-FAPP1 was localized primarily to the plasma membrane in the tips of growing root hairs (Figures 5D and 6A; see Supplemental Movie 3 online), although some internal membranes appeared to be lightly labeled by the EYFP-FAPP1 protein as well (Figure 5D, arrowheads). By contrast, in rhd4-1 root hairs, EYFP-FAPP1 accumulated predominantly on internal membrane compartments within the tip. We observed little accumulation of EYFP-FAPP1 in the plasma membrane at the tips of root hair cells (Figures 5G and 6B; see Supplemental Movie 4 online).

Figure 6.

Figure 6.

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EYFP-FAPP1 Localization in Wild-Type and rhd4-1 Root Hairs.

Arabidopsis seedlings expressing EYFP-FAPP1 were imaged by time-lapse fluorescence microscopy. Beginning, middle, and end frames are shown. Bars = 10 μm.

(A) Wild-type root hair. These frames are from Supplemental Movie 3 online.

(B) rhd4-1 root hair. These frames are from Supplemental Movie 4 online.

To better demonstrate the difference between EYFP-FAPP1 localization in wild-type and rhd4-1 root hairs, we studied the localization of the fusion protein in root hair cross sections. Cross sections at various positions within the root hair tip were obtained from z-stacks using Imaris software, and ImageJ was used to quantify the pixel intensity along a vertical line through the center of the cross section as described in Methods. In wild-type root hairs (Figure 5D), EYFP-FAPP1 was localized primarily to the plasma membrane, and in cross section and by quantification, the pixel intensity shows two peaks corresponding to the two edges of the root hair (Figures 5E and 5F). However, in rhd4-1 root hairs, EYFP-FAPP1 was found on membrane compartments throughout the root hair (Figure 5H, arrowheads); therefore, in cross section and by quantification, the pixel intensity is variable (Figures 5H and 5I). We have observed FAPP1 localization in multiple transgenic rhd4 lines and in lines where EYFP-FAPP1 was crossed into the rhd4 background; all lines we observed show the same FAPP1 localization. These results strongly support the conclusion that mutation of the RHD4 PI(4)P phosphatase results in altered subcellular distribution of PI(4)P within the root hair cell, while distribution of PI(3)P remains unchanged.

The Actin Cytoskeleton Is Altered in rhd4-1 Epidermal Cells

Yeast Sac1p mutants are known to have defects in actin cytoskeleton organization (Foti et al., 2001), and mutation of At SAC1 also results in altered actin cytoskeleton organization in fiber and pith cells of elongating stems (Zhong et al., 2005). We were interested in determining whether mutation of the gene encoding the RHD4 phosphatase caused similar defects in actin organization. The GFP-FABD2 actin filament marker (Voigt et al., 2005a) was used to observe the actin cytoskeleton in wild-type and rhd4-1 root hairs and epidermal cells. Actin filament organization does not appear to be altered in rhd4-1 root hairs (Figures 7C and 7D) compared with wild-type root hairs (Figures 7A and 7B). However, in root epidermal cells, more actin patches were observed and filaments were thinner in rhd4-1 cells (Figure 7F) compared with wild-type cells (Figure 7E).

Figure 7.

Figure 7.

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GFP-FABD2 Localization in rhd4-1 Root Hairs and Epidermal Cells.

Arabidopsis seedlings expressing GFP-FABD2 (for localization of F-actin filament bundles) were imaged by confocal microscopy, and z-stack projections are shown. Bars = 20 μm.

(A) and (B) Localization in expanding wild-type root hairs. F-actin filament bundles are found throughout the root hair.

(C) and (D) Localization in expanding rhd4-1 root hairs. F-actin filaments do not appear to be disturbed.

(E) Localization in wild-type root epidermal cells. A meshwork of F-actin is observed.

(F) Localization in rhd4-1 root epidermal cells. A meshwork of F-actin is observed similar to the wild type; however, more actin patches are observed.

RHD4 Colocalizes with RabA4b in the Tips of Root Hairs

The altered localization of PI(4)P observed on internal membranes in rhd4-1 root hairs implied that wild-type RHD4 phosphatase activity limited the levels of this phosphoinositide in membranes of these internal compartments. It is known that yeast Sac1p is localized to both the endoplasmic reticulum (ER) and Golgi compartments (Konrad et al., 2002), and mammalian Sac1 is predominantly localized to the ER (Nemoto et al., 2000). We were therefore interested in determining RHD4 subcellular localization in plants, specifically within root hairs. An EYFP-RHD4 fusion protein was constructed and transformed into Arabidopsis under control of the native RHD4 promoter. EYFP-RHD4 was functional, as it was capable of rescuing the rhd4-1 mutant phenotype (Figures 3C and 3D, Table 1).

Intriguingly, subcellular EYFP-RHD4 was localized to the tips of growing root hairs (Figures 3C and 8A), showing a strikingly similar distribution to that observed for EYFP-RabA4b (Figure 8B). To compare the localization of RHD4 and RabA4b to Golgi and ER compartments, YFP-Golgi (G-yk) and YFP-ER (ER-yk) were obtained as markers for the Golgi and ER (Nelson et al., 2007). In root hairs, the tip-restricted localization pattern of RHD4 and RabA4b is distinct from YFP-Golgi (Figure 8C) and YFP-ER (Figure 8D), which are distributed throughout the cytoplasm of the root hair cell. Localization in root epidermal cells is also shown for EYFP-RabA4b (Figure 8E), YFP-Golgi (Figure 8F), and YFP-ER (Figure 8G). Some cells contain bright fusiform bodies that are local extensions of the ER (Nelson et al., 2007). However, EYFP-RHD4 is not expressed highly enough to image the localization in these cells; therefore, its epidermal cell localization could not be compared.

Figure 8.

Figure 8.

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EYFP-RHD4 Is Localized to the Tips of Growing Root Hairs.

(A) to (D) Arabidopsis seedlings expressing various YFP fusion proteins were imaged by confocal microscopy. A medial fluorescence image, the corresponding differential interference contrast (DIC) image, and an overlay of these images are shown (columns, left to right). Bars = 10 μm.

(A) EYFP-RHD4 was expressed by its native promoter in rhd4-1 Arabidopsis plants and is localized to the tips of growing root hairs.

(B) EYFP-RabA4b is localized to a post-Golgi secretory compartment at the tips of growing root hairs.

(C) G-yk (a YFP-Golgi maker) is localized to the Golgi compartment throughout growing root hairs.

(D) ER-yk (a YFP-ER marker) is localized to the ER throughout growing root hairs.

(E) to (G) Arabidopsis seedlings expressing YFP fusion proteins were imaged by confocal microscopy. Inset: higher magnification. Z-stack projections are shown. Bars = 20 μm.

(E) EYFP-RabA4b localization in root epidermal cells.

(F) G-yk localization in root epidermal cells.

(G) ER-yk localization in root epidermal cells.

(H) to (J) Arabidopsis seedlings expressing EYFP-RHD4 were fixed, processed for immunolocalization of anti-RabA4b antibody, and analyzed by confocal microscopy. Medial sections are shown. Bars = 10 μm.

(H) and (I) RabA4b colocalizes with EYFP-RHD4 at the tips of root hairs. EYFP-RHD4 (green) and anti-RabA4b (red) are localized to overlapping tip-localized membrane compartments (yellow).

(J) Tip localization of anti-RabA4b is specific, as no tip-localized fluorescence was observed when anti-RabA4b primary antibody was left out.

To further determine whether RHD4 and RabA4b were localized to the same compartment in root hairs, we observed the localization of the anti-RabA4b antibody (Preuss et al., 2004) in fixed root hairs from transformed plants expressing EYFP-RHD4. These compartments colocalize at the tips of fixed root hairs (Figures 8H and 8I), and no tip-localized fluorescence was detected when anti-RabA4b antibodies were left out (Figure 8J). We have previously demonstrated that RabA4b labels budding profiles emerging from plant TGN compartments and post-Golgi vesicular structures (Preuss et al., 2006). Furthermore, both RabA4b and its effector protein, PI-4Kβ1, colocalize in the tips of root hair cells on membranes that are distinct from plant Golgi compartments. Based on these results, we concluded that, unlike in yeast and mammals, the Sac1p-like PI(4)P phosphatase localizes primarily to post-Golgi secretory compartments and participates in limiting the levels of PI(4)P in these membranes.

DISCUSSION

In Arabidopsis, polarized root hair growth is tightly associated with the polar accumulation of membrane compartments labeled by the RabA4b GTPase (Preuss et al., 2004). Furthermore, the RabA4b effector protein PI-4Kβ1 is required for proper control of polarized root hair expansion (Preuss et al., 2006). A number of root hair development mutants have been described with defects in root hair initiation and development. In this study, we further characterize one of these developmental mutants, rhd4-1, which displays shorter root hairs that sporadically form bulges as they expand (Schiefelbein and Somerville, 1990; Galway et al., 1999). In rhd4-1 mutants, RabA4b-labeled compartments displayed altered distributions, with loss of tip localization that appeared to be associated with formation of root hair bulges (Preuss et al., 2004).

Here, we describe further analysis of the dynamics of EYFP-RabA4b localization in rhd4-1 mutant root hairs and determine RHD4 to be a Sac1p-like phosphoinositide phosphatase. Furthermore, we have shown that RHD4 has PI(4)P phosphatase activity based on both in vitro and in vivo analyses. We found that PI(4)P was primarily associated with a tip-localized plasma membrane domain in wild-type plants, but this distribution was altered in rhd4-1 mutant root hairs, accumulating significantly on internal membrane compartments. This qualitative difference may reflect the effect of increased amounts of PI(4)P in rhd4-1 root tissues revealed by the in vivo labeling study. Also, RHD4 colocalized with RabA4b in a trans-Golgi network compartment at the tips of root hairs. These results, along with the previous determination that PI-4Kβ1 is required for proper control of root hair tip growth, demonstrate that regulation of PI(4)P levels plays a central role in polarized growth during root hair development.

Yeast SAC1 was initially characterized as a suppressor of a conditional-lethal temperature-sensitive actin mutation (Novick et al., 1989), and it was later identified as a phosphoinositide phosphatase (Hughes et al., 2000; Foti et al., 2001). Subsequently, Sac1p, an essential gene in yeast, has been shown to regulate PI(4)P levels and to play important roles in the Golgi apparatus, actin cytoskeleton dynamics, and other cellular functions (Hughes et al., 2000; Foti et al., 2001). Studies in Drosophila melanogaster have shown that mutation of Sac1 results in disruption of JNK mitogen-activated protein kinase signaling, resulting in embryo lethality (Wei et al., 2003). It is intriguing that, in plants, the rhd4-1 null allele has defective root hairs but no other observed morphological defects. This may suggest that RHD4 phosphatase activity is particularly important in cells in which differentiation involves significant levels of polarized secretion, such as root hair cells.

One possible reason for the limited defects of a SAC phosphatase null mutant in Arabidopsis is that the genome contains nine SAC genes. Mutation of the RHD4 PI(4)P phosphatase results in aberrant root hair development, and no other phenotypes have been observed with this mutant, suggesting that its close family members SAC6 and 8 may have a redundant function in PI(4)P regulation in other parts of the plant. This may explain why, upon deletion of the RHD4 gene, we observed an approximate twofold increase in PI(4)P in rhd4-1 mutant root tissue, as opposed to the 8- to 10-fold increase observed in yeast Sac1p mutants that contain only a single SAC1 gene (Guo et al., 1999; Hughes et al., 2000).

It is important to note that RHD4 is the only gene of this family (At SAC6, 7, and 8) expressed in root hairs, according to a promoter-GUS study (Despres et al., 2003), and this may explain why only a root hair phenotype is observed in rhd4 mutants. When expressed under the control of its native promoter, EYFP-RHD4 is localized to the tips of root hairs and colocalized with EYFP-RabA4b, suggesting that RHD4 is also localized to this post-Golgi compartment. By contrast, in yeast, Sac1p has been localized to both ER and Golgi membranes (Konrad et al., 2002), while rat Sac1 was localized to the ER (Nemoto et al., 2000).

Previously, Despres et al. (2003) found RHD4 was colocalized to the ER when a C-terminal GFP fusion of the protein was expressed in tobacco (Nicotiana tabacum) BY2 cells. However, it is possible that overexpression of this integral membrane protein caused mislocalization or that BY2 cells are an inappropriate tissue for observing the localization of this fusion protein. Alternatively, perhaps this C-terminal GFP fusion could not be trafficked properly, whereas our N-terminal EYFP fusion is known to be functional, as demonstrated by complementation of the rhd4-1 phenotype. It also is possible that, in cells undergoing high levels of polarized secretion such as root hairs and pollen tubes, RHD4 is found in subcellular locations unique from their yeast or mammalian homologs to specifically influence membrane trafficking required for polarized secretion.

Earlier transmission electron microscopy studies found that rhd4-1 root hairs had localized cell wall thickenings that were thought to form during root hair bulging (Galway et al., 1999). These thickenings could occur in rhd4-1 root hairs when an overaccumulation of PI(4)P leads to increased vesicle production and/or spurious fusion at the tips of root hairs, resulting in increased delivery of cell wall materials and in root hair bulging. Interestingly, in yeast, loss of Sac1p leads to increased trafficking of chitin synthase to the plasma membrane, and Sac1p has been identified as an antagonist of Pik1p, the yeast homolog of PI-4Kβ1 (Schorr et al., 2001). In plants, loss of PI-4Kβ activity results in compromised control of root hair tip growth (Preuss et al., 2006), as does the loss of RHD4 PI(4)P phosphatase activity.

Taken together, we propose that the defect in proper root hair tip growth upon mutation of either of these components is due to loss of proper regulation of PI(4)P levels within these cells. We found that EYFP-RHD4 was localized to subcellular compartments in the tips of root hairs (Figure 8), where we propose it functions to regulate levels of PI-4Kβ1-generated PI(4)P, and that it normally restricts the levels of PI(4)P to vesicles at the extreme tips of growing root hairs. In the rhd4-1 mutant, loss of the phosphatase activity would result in release of this inhibition, and PI(4)P accumulation on these subcellular membranes could occur (Figures 5G). This may cause an increased accumulation of PI(4)P-labeled vesicles, resulting in wider root hairs, and in extreme cases bulge formation may occur.

Our results are similar to results for PLC3 regulation of PI(4,5)P2 in pollen tubes in which PLC3 is targeted to the flanks of pollen tube tips, but not to the apex, where it functions to prevent overaccumulation of PI(4,5)P2 outside of the apex, thus maintaining PI(4,5)P2 at the tips (Helling et al., 2006). Also of interest is a recent study in animal cells demonstrating that enrichment of PI(4,5)P2 on the apical plasma membrane of epithelial cells is due to the localization of the PI(3,4,5)P3 phosphatase PTEN (Phosphatase and Tensin homolog on chromosome 10) to this subcellular domain (Martin-Belmonte et al., 2007). This study suggests that PI(4,5)P2 acts as a signal to differentiate the apical membranes from basolateral membranes in these cells, a process that had not previously been understood.

Additionally, Nishio et al. (2007) have recently found that SHIP1, a PI(3,4,5)P3 phosphatase, functions to regulate the localized accumulation of PI(3,4,5)P3 required for polarization and motility in mouse neutrophils. These observations and the results we present here demonstrate that phosphoinositide signals have an effect on cellular morphogenesis. Therefore, regulating not only the levels but also the locations where various phosphoinositides accumulate within cells during development may be a conserved mechanism to generate spatial cues that influence the establishment and maintenance of polarity.

In conclusion, we demonstrated that RHD4 encodes a Sac1p-like phosphoinositide phosphatase that regulates a pool of PI(4)P, which ultimately influences root hair development. We found that both PI-4Kβ1 and RHD4 were localized to RabA4b-labeled membranes in the tips of root hairs, where we propose that they function to maintain PI(4)P levels required for proper polarized secretion.

METHODS

Visualization of EYFP-RabA4b Localization

Wild-type and rhd4-1 Arabidopsis thaliana seedlings expressing EYFP-RabA4b were grown on 0.25× MS medium + 0.3% phytagel. Seven- to 10-d-old seedlings were transferred to a slide chamber through which media were exchanged by peristaltic pump, as previously described (Preuss et al., 2004). EYFP fluorescence was observed with a Nikon Eclipse E600 microscope with a ×40 Plan Apo lens (numerical aperture 1.2) with frames collected every 30 s. Relative length was defined as the change in root hair length from time 0 and length 0 μm. The fluorescent signal located within the proximal 7% of the length of the root hair was defined as tip fluorescence, and this was presented as a percentage of the total fluorescence detected within the entire root hair.

Map-Based Cloning

The rhd4-1 mutant (Columbia ecotype) was crossed with Arabidopsis ecotype Landsberg erecta to generate F2 mapping plants. Coarse mapping was done with publicly available CAPS and SSLP genetic markers. Fine mapping of rhd4-1 was done with CAPS and SSLP markers developed based on Landsberg sequence information from the Cereon Arabidopsis database (Jander et al., 2002).

For rhd4-1 complementation, the pEarleyGate302 vector (Earley et al., 2006) was used. The wild-type RHD4 cDNA was generated by RT-PCR from wild-type Columbia cDNA template using the primers XbaI RHD4 forward (5′-TCTAGAATGGAGACGGTTGATTCTCGG-3′) and PacI RHD4 reverse (5′-TTAATTAATCAGCCTCTGGGCTTGTG-3′) and inserted into pEarleyGate302 using restriction sites for XbaI and PacI. The RHD4 upstream sequence was cloned using the primers RHD4 Gateway promoter forward (5′-CACCAATATTATGTTTTCACAAATAAGGAAAGTTTCC-3′) and RHD4 Gateway promoter reverse (5′-ACTAAAATCAAACAAGAGACAAAAGAAGATTC-3′) and was recombined by Clonase reaction into pEarleyGate302.

rhd4-1 plants were transformed with Agrobacterium tumefaciens by the floral dip method (Clough and Bent, 1998). Transgenic plants were selected on 0.25× MS + Basta (glufosinate ammonium; Fisher) and carried to the T2 generation to verify complementation of the rhd4-1 phenotype.

Characterization of Additional RHD4 Alleles

The rhd4-2 EMS allele was obtained from J. Schiefelbein (University of Michigan, Ann Arbor, MI). The RHD4 insertion mutants were obtained from the SALK T-DNA collection (rhd4-3 is SALK_079231 and rhd4-4 is SALK_092575; ABRC) (Alonso et al., 2003). Wild-type and mutant seedlings were grown in 0.25× MS + 0.3% phytagel, transferred to a slide, and imaged using a Nikon Eclipse E600 microscope with a ×40 Plan Apo lens (numerical aperture 1.2). The T-DNA insertion site in each line was confirmed by sequencing (primers LBA1 5′-TGGTTCACGTAGTGGGCCATCG-3′, rhd4-3 5′-GATCCAACACGTTCACTCTCAG-3′, and rhd4-4 5′-TCCATGAAGCTCATCGGTTG-3′). Root hair length was quantified by measuring root hairs with Image J and calculating the average and standard deviation. Significance was determined using a two-sample t test assuming unequal variances.

RT-PCR

Wild-type Columbia Arabidopsis seedlings were grown at 22°C with 16 h of light for 10 d. Total RNA was extracted using the RNeasy kit (Qiagen), and cDNA was made according to TITANIUM one-step RT-PCR kit instructions (Clontech) with the primers RHD4 RT forward (5′-TCTAGAATGGAGACGGTTGATTCTCGG-3′) and RHD4 RT reverse (5′-TGGAGAACTCGGTCTCCATC-3′). Generation of β-tubulin cDNA from the same template RNA was used to equalize levels of cDNA (β-tubulin forward 5′-CCTGATAACTTCGTCTTTGG-3′ and β-tubulin reverse 5′-GTGAACTCCATCTCGTCCAT-3′). The amplification program consisted of 30 s at 94°C, 30 s at 65°C, and 1 min at 68°C for 35 cycles, followed by a 2-min extension at 68°C.

Protein Purification

Truncated cDNAs of RHD4 and the rhd4-2 allele of RHD4 (lacking the C-terminal transmembrane domains) were generated by RT-PCR for expression as GST fusion proteins with primers SmaI GST-RHD4 forward (5′-CCCGGGGATGGAGACGGTTGATTCTCGG-3′) and XhoI GST-RHD4 truncated reverse (5′-CTCGAGTCACCCTTGCAAGAGATCAATTGC-3′). These cDNAs were then inserted in frame into the pGEX-4T1 expression vector using restriction sites for SmaI and XhoI. The fusion proteins were expressed in Escherichia coli Rosetta cells and purified with glutathione sepharose beads. Purified protein was eluted with 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 15 mM glutathione, 2 mM DTT, and 0.25% β-d-octylglucoside.

The cDNA encoding the 2XFYVE PH domain was generated using pGFP-2XFYVE as a template, with primers BamHI GST-FYVE forward (5′-GGATCCGAAAGTGATGCCATGTTC-3′) and XhoI GST-FYVE reverse (5′-GAGCTCTGCCTTCTTGTTCAGCTG-3′). The cDNA encoding the FAPP1 PH domain was generated using pHAN-hFAPP1 (gift from Marino Zerial) as a template, with primers NdeI GST-FAPP1 forward (5′-GGGAATTCCATATGATGGAGGGGGTGTTGTAC-3′) and KpnI GST-FAPP1 reverse (5′-GGGGTACCTCAAGTCCTTGTATCAGTCAAACATGC-3′). These cDNAs were then inserted in frame into the pET41 expression vector with the restriction sites BamHI and XhoI (2XFYVE) and NdeI and KpnI (FAPP1). The fusion proteins, along with the pET41 GST control, were expressed in E. coli Rosetta cells (GST-FAPP1 and GST) or BL21 cells (GST-2XFYVE) and purified with glutathione sepharose beads. Purified protein was eluted with 50 mM Tris, pH 8.0, 100 mM NaCl, 15 mM glutathione, and 5 mM β-mercaptoethanol.

Phosphatase Activity Assay

Purified recombinant GST, GST-RHD4, and GST-rhd4-2 were used for a phosphoinositide phosphatase activity assay. diC16 phospholipid substrates (Echelon) were dissolved in 1:2:0.8 CHCl3:MeOH:H2O with the aid of sonication. The phosphatase activity of recombinant proteins was measured by mixing 2.5 μg of protein in reaction buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 10 mM KCl, 100 mM NaCl, 0.25% β-d-octylglucoside, and 1 mM phenylmethylsulfonyl fluoride) with 100 μM phospholipid substrates in a 50-μL reaction volume and incubated at 30°C for 50 min, agitating every 10 min. The reactions were stopped by the addition of 100 μL Biomol Green (Biomol International), incubated at room temperature for 30 min, and the free phosphate released from the substrates was measured at OD620 and determined by comparison to NaPO4 standards.

In Vivo Labeling of Phosphoinositides

Wild-type and rhd4-1 mutant Arabidopsis seedlings were grown in 0.25× MS containing 10 μM reduced myo-inositol for 2 weeks. Two-week-old plants were radiolabeled with myo-2-[3H] inositol at a concentration of 50 μCi/mL. Labeling was done in 1 mL in wells of a 24-well plate for 40 h on a gyratory shaker at 22°C.

Lipids were extracted using the protocol of Hama et al. (2000). Briefly, shoot and root tissues were separated, and root tissues were fixed in 5% trichloroacetic acid and incubated on ice for 1 h. Tissue was washed five times with 10 mL of water, suspended in 0.5 mL of water in a tissue grinder, and homogenized with the addition of 0.75 mL 95% ethanol:diethyl ether:pyridine (15:5:1 [v/v]). The homogenate was incubated at 57°C for 30 min. Cell debris was removed by centrifugation, and the supernatant was dried under N2.

Dried lipids were resuspended in 2 mL of water:methylamine (3:7, methylamine is 40% [v/v] in ethanol) by water bath sonication and vortex, incubated for 50 min at 53°C, and dried under N2. Deacylated lipids were suspended in 0.5 mL of water by water bath sonication and extracted with 1 mL of water-saturated n-butanol:petroleum ether:ethyl formate (20:4:1) four times. The aqueous phase was dried under N2.

Deacylated lipids were analyzed by anion exchange HPLC as previously described (Bonangelino et al., 2002).

Lipid Binding Assays

Lipid binding assays were performed using PIP Strips (Echelon). PIP strips were blocked in 1× TBS, 0.1% Tween 20, and 5% fatty acid free BSA and then incubated with 1 nM protein at 4°C overnight (either GST, GST-FAPP, or GST-2XFYVE). Next, the PIP strips were washed with 1× TBS + 0.1% Tween 20, incubated with anti-GST antibody (1:2000 in blocking solution) for 1 h at room temperature, washed with 1× TBS + 0.1% Tween 20, and then incubated with anti-rabbit HRP secondary antibody (1:2500) for 1 h at room temperature. Finally, the PIP strips were washed with 1× TBS + 0.1% Tween 20 before developing with enhanced chemiluminescent reagent.

Localization of PI Pools

pCAMBIA-35S-EYFP-FAPP1 vectors were constructed by subcloning the EYFP-FAPP1 fusion from the vector pMON999YFP-PHFAPP into pCAMBIA (CAMBIA) using restriction sites NcoI and BamHI. Wild-type and rhd4-1 transgenic EYFP-FAPP1 plants were generated with Agrobacterium by the floral dip method (Clough and Bent, 1998).

Wild-type and rhd4-1 EYFP-FAPP1 seedlings were observed on a confocal microscope (Zeiss LSM 510) with a ×40 Apochromat lens (numerical aperture 1.2). Z-stack sections of root hairs were taken, and three-dimensional projections from these stacks were used for the final images. For Figure 5D, there are 25 slices, taken 0.66 μm apart; for Figure 5G, there are 38 slices, taken 0.66 μm apart. Imaris software (Bitplane) was used to obtain cross sections of root hairs from the z-stacks. ImageJ (software developed by the National Institutes of Health and available from http://rsb.info.nih.gov/ij) was used to quantify the pixel intensity in the root hair cross sections.

GFP-2XFYVE Arabidopsis seed was a gift from F. Baluska (University of Bonn, Bonn, Germany). Seedlings were observed on a confocal microscope (Leica confocal system TCS SP2) with a ×63 Plan Apo lens (numerical aperture 1.2). Z-stacks of root hairs were taken, and three-dimensional projections from these stacks were used for the final images. For Figure 5B, there are 12 slices, taken 1.0 μm apart; for Figure 5C, there are 22 slices, taken μm apart.

Localization of EYFP-RHD4 and GFP-FABD2

The RHD4 cDNA was generated by RT-PCR from wild-type Columbia cDNA using the primers RHD4 forward (5′-GTCGACATGGAGACGGTTGATTCTC-3′) and RHD4 reverse (5′-TCTAGATCAGCCTCTGGGCTTGTG-3′) and inserted into the pCAMBIA vector (CAMBIA) using restriction sites for SalI and XbaI. The RHD4 upstream sequence was generated by PCR from wild-type Columbia genomic DNA using the primers EcoRI RHD4 promoter forward (5′-GAATTCAATATTATGTTTTCACAAATAAGGAAAGTTTCC-3′) and NcoI RHD4 promoter reverse (5′-CCATGGACTAAAATCAAACAAGAGACAAAAGAAGATTC-3′). This sequence was inserted into pCAMBIA using restriction sites for EcoRI and NcoI to drive expression of EYFP-RHD4. Wild-type and rhd4-1 EYFP-RHD4 plants were generated with Agrobacterium by the floral dip method (Clough and Bent, 1998).

G-yk and ER-yk Arabidopsis were a gift from A. Nebenfuhr (University of Tennessee, Knoxville, TN).

GFP-FABD2 Arabidopsis seed were a gift from F. Baluska (University of Bonn, Bonn, Germany). Pollen from wild-type GFP-FABD2 plants was crossed to rhd4-1 stigmas to create rhd4-1 GFP-FABD2 plants.

Seedlings were grown on 0.25× MS + 0.3% phytagel, and 7- to 10-d-old seedlings were observed on a confocal microscope (Zeiss LSM 510) with a ×40 Apochromat lens (numerical aperture 1.2). For images in Figure 7 and epidermal cell images in Figure 8, z-stacks of root hairs and epidermal cells were taken and three-dimensional projections were used for the final images. For Figure 7A, 25 slices were imaged; for Figure 7B, 22 slices; for Figure 7C, 25 slices; for Figure 7D, 38 slices; for Figure 7E, 47 slices; for Figure 7F, 43 slices. All slices were taken 1.06 μm apart. For Figure 8E, 26 slices were imaged; for Figure 8F, 22 slices; for Figure 8G, 10 slices. All slices were taken 1.0 μm apart. For root hair images in Figure 8, medial sections are shown.

Immunolocalization

EYFP-RHD4 Arabidopsis seedlings were processed for immunolocalization as previously described (Preuss et al., 2006). Primary anti-RabA4b antibody (Preuss et al., 2004) was used at a concentration of 1:100, and secondary anti-rabbit Cy3 was used at a concentration of 1:200. After primary and secondary antibody incubations, slides were mounted with Mowiol (Calbiochem). Samples were observed on a confocal microscope (Zeiss LSM 510) with a ×40 Apochromat lens (numerical aperture 1.2). Medial sections were used for the final images. Images were taken at the same laser power for accurate comparison of Cy3 signal.

Accession Numbers

The RHD4/SAC7 Arabidopsis Genome Initiative number is At3g51460, and the GenBank accession number is AY227250. Accession numbers for the mutant alleles of rhd4 shown in Figures 2A and 3B are as follows: The Arabidopsis Information Resource accession 1945311 or Germplasma CS2261 (rhd4-1), Germplasma CS16302 (rhd4-2), SALK_079231 (rhd4-3), and SALK_092575 (rhd4-4). The GenBank accession numbers for the sequences shown in Figure 2B are X51672 (Sac1p), NM_014937 (hSac2), NM_053798 (rSac1), and NM_003895 (Synaptojanin I).

Supplemental Data

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

  • Supplemental Figure 1. rhd4-1 Mutant Transcript Sequence.

  • Supplemental Movie 1. EYFP-RabA4b Is Localized to the Tips of Wild-Type Root Hairs.

  • Supplemental Movie 2. EYFP-RabA4b Localization Is Altered in rhd4-1 Root Hairs.

  • Supplemental Movie 3. EYFP-FAPP1 Is Localized to the Plasma Membrane at the Tips of Wild-Type Root Hairs.

  • Supplemental Movie 4. EYFP-FAPP1 Localization Is Altered in rhd4-1 Root Hairs.

  • Supplemental Movie Legends.

Supplementary Material

[Supplemental Data]
plntcell_tpc.107.054304_index.html (1.3KB, html)

Acknowledgments

We thank John Schiefelbein for sharing additional rhd4 EMS mutant lines (rhd4-2), Frantisek Baluska for sharing GFP-2XFYVE and GFP-FABD2 transformed Arabidopsis lines and the pGFP-2XFYVE plasmid, Andreas Nebenfuhr for sharing ER-yk and G-yk transformed Arabidopsis lines, Marino Zerial for the pHAN-hFAPP plasmid, Jonathan Markham for technical assistance with lipid extraction techniques, Amy Steward and Heather Bonner for technical assistance with FAPP1 work, and Howard Berg for help with various microscopy work. We thank Ralph Quatrano and his lab for bench space, reagents, and valuable discussion. We also thank Lois Weisman for technical help and input regarding in vivo analysis of PI-4P levels. This work was supported by the Department of Energy (DE-FG02-03ER15412 to E.N.).

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: Erik Nielsen (nielsene@umich.edu).

[W]

Online version contains Web-only data.

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