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. 2006 Mar 27;172(7):991-8.
doi: 10.1083/jcb.200508116.

A role for the RabA4b effector protein PI-4Kbeta1 in polarized expansion of root hair cells in Arabidopsis thaliana

Mary L Preuss  1 , Aaron J Schmitz, Julie M Thole, Heather K S Bonner, Marisa S Otegui, Erik Nielsen
Affiliations

A role for the RabA4b effector protein PI-4Kbeta1 in polarized expansion of root hair cells in Arabidopsis thaliana

Mary L Preuss et al. J Cell Biol. .

Abstract

The RabA4b GTPase labels a novel, trans-Golgi network compartment displaying a developmentally regulated polar distribution in growing Arabidopsis thaliana root hair cells. GTP bound RabA4b selectively recruits the plant phosphatidylinositol 4-OH kinase, PI-4Kbeta1, but not members of other PI-4K families. PI-4Kbeta1 colocalizes with RabA4b on tip-localized membranes in growing root hairs, and mutant plants in which both the PI-4Kbeta1 and -4Kbeta2 genes are disrupted display aberrant root hair morphologies. PI-4Kbeta1 interacts with RabA4b through a novel homology domain, specific to eukaryotic type IIIbeta PI-4Ks, and PI-4Kbeta1 also interacts with a Ca2+ sensor, AtCBL1, through its NH2 terminus. We propose that RabA4b recruitment of PI-4Kbeta1 results in Ca2+-dependent generation of PI-4P on this compartment, providing a link between Ca2+ and PI-4,5P2-dependent signals during the polarized secretion of cell wall components in tip-growing root hair cells.

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Figures

Figure 1.
Figure 1.
RabA4b interacts specifically with PI-4Kβ1. (A) Yeast two-hybrid interaction of PI-4Kβ1Δ1-421 with active GTP bound RabA4b (Q), but not inactive GDP bound RabA4b (S), was detected on high-stringency media (−HisTrpLeu [HTL] + 3-AT). No interaction was observed with vacuolar RabG3c; members of two other plant PI-4K classes, PI-4Kα1 and -4Kγ6; or the plant PI-3K AtVPS34. Presence of prey and/or bait vectors were monitored by growth in absence of tryptophan and leucine (−TL) or tryptophan (−T), respectively. (B) LKU, repetitive, NH, and catalytic domains are indicated. Deletion fragments of PI-4Kβ1 were constructed to determine the binding site of RabA4b. (C) Yeast two-hybrid interaction was seen between active RabA4b (Q) and PI-4Kβ1 fragments containing the NH domain (Δ1-421 and NH) on selective media (−HisTrpLeu + 3-AT). No interaction was observed between RabA4b and other PI-4Kβ1 domains (LKU, repetitive, and catalytic). Surprisingly, full-length PI-4Kβ1 did not interact with RabA4b in the yeast two-hybrid system. (D) PI-4Kβ1 and its NH domain, but not PI-4Kγ6 and AtVPS34, were selectively recruited by active GST-RabA4b–GTPγS, confirming the specificity of the PI-4Kβ1–RabA4b interaction.
Figure 2.
Figure 2.
PI-4Kβ1 colocalizes with EYFP-RabA4b on tip-localized compartments in root hair cells. (A) Anti-PI4Kβ1 recognized an ∼125-kD protein band (arrow) present in postnuclear supernatant (PNS) and membrane fractions (Pel) but not soluble fractions (Sol). The 40-kD band in soluble fractions is present only in green tissues and is not detected in isolated root protein fractions (not depicted). (B–E) A. thaliana seedlings were fixed, processed for immunofluorescence, and analyzed by laser confocal microscopy to detect localization of EYFP-RabA4b or EGFP-GmManI fluorescence (green) and PI-4Kβ1 (red). (B) PI-4Kβ1 was tip localized in root hairs. (C) PI-4Kβ1 localization (red) was distinct from Golgi membranes containing EGFP-GmManI (green). (D and E) Detection of tip-localized PI-4Kβ1 compartments was specific, as no tip-localized fluorescence was observed if only anti–PI-4Kβ1 primary antibodies were left out (D) or no antibodies were used (E). (F) Only background fluorescence was observed in root hairs of the PI-4Kβ1/β2 double mutant.
Figure 3.
Figure 3.
PI-4Kβ1/β2 function is essential for normal A. thaliana growth and root hair development. (A) Sequencing of β1-1 and β2-1 T-DNA insertion sites confirmed the positions of the T-DNA inserts within PI-4Kβ1 (intron 7) and -4Kβ2 (intron 8) in these two lines. (B) Total RNA was extracted from seedlings homozygous for a T-DNA insertion in PI-4Kβ1 (β1-1/β1-1), PI-4Kβ2 (β2-1/β2-1), or WT and used for RT-PCR. PI-4Kβ1 transcript was not detected in β1-1/β1-1, and PI-4Kβ2 transcript was not detected in β2-1/β2-1. Tubulin was amplified as a loading control. (C) Anti–PI-4Kβ1 antibodies detected an ∼125-kD protein band in immunoblots of total protein extracts from WT seedlings but not from PI-4Kβ1/PI-4Kβ2 double-mutant seedlings (β1-1/β1-1/β2-1/β2-1). (D) Double mutants (β1-1/β1-1/β2-1/β2-1) were smaller than WT plants. Plants homozygous for the PI-4Kβ1 insertion but heterozygous for the PI-4Kβ2 insertion (β1-1/β1-1/WT/β2-1) were intermediate in size. (E) Double-mutant root hairs were shorter and growth was disorganized compared with WT. (F) Double-mutant root hairs were classified into six classes based on their phenotype (normal, branched, bulged, popsicle, jagged, and wavy). Representative images of each class are shown. (G, left) WT root hairs were longer than in double mutants. wt, n = 817; β1/β2, n = 797. (right) Percentages of each root hair class were determined in mutant and WT plants. Most WT root hairs were normal in appearance (>90%). However, double mutants had <50% normal root hairs and much higher percentages of each class of deformed root hairs. Root hairs were counted from 19 WT and 21 β1-1/β1-1/β2-1/β2-1 plants. Error bars indicate SD.
Figure 4.
Figure 4.
RabA4b-labeled membranes have altered morphologies in PI-4Kβ1/β2 double mutants. (A–F) High-pressure frozen/freeze-substituted root tip cells from WT or PI-4Kβ1/β2 double-mutant A. thaliana plants expressing EYFP-RabA4b were processed for EM analysis. (A) Golgi (G) and TGN compartments from WT cells. Note distinct TGN budding profiles (arrows). (B–D). In PI-4Kβ1/β2 double mutants, TGN were often aberrant, with lighter staining and clustered budding profiles (arrowheads). (E and F) Affinity-purified anti-RabA4b antibodies labeled TGN budding profiles in both WT (E, arrows) and PI-4Kβ1/β2 double mutants (F, arrowheads). (G) The numbers of distinct TGN budding profiles associated with each sample (defined as a section containing at least one discernible Golgi stack and associated TGN) were counted. WT averaged seven to nine TGN budding profiles per sample (n = 51), whereas only one to three TGN budding profiles were usually observed per sample in double mutants (n = 49).
Figure 5.
Figure 5.
The Ca2+ sensor AtCBL1 interacts with NH2-terminal domains of PI-4Kβ1. (A) Yeast two-hybrid interaction between AtCBL1 and the NH2-terminal PI-4Kβ1 fragment (ΔC567-1121; Fig. 1 B) was observed on high-stringency media (−HisTrpLeu [HTL] + 3-AT). (B–D) Disruption of tip-focused Ca2+ gradient in root hairs abolished growth and tip-localized EYFP-RabA4b. (B) EYFP-RabA4b fluorescence was visualized in root hairs using time-lapse fluorescence microscopy. Upon treatment with the Ca2+ ionophore A23187 (20-min time point), root hair elongation was rapidly inhibited. This correlated with loss of EYFP-RabA4b tip localization and observation of EYFP-RabA4b along the entire root hair (24–30-min time points). When A23187 was washed out, all EYFP-RabA4b fluorescence was lost from the root hair. Neither EYFP-RabA4b tip localization nor root hair tip growth occurred after A23187 washout. The length of the root hair (C) and the percentage of tip fluorescence (D) were measured over time.
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