Article Figures & Data
Figures
- 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 = 100 μm.
(E) and (F) Root cross sections. Bars = 25 μm.
(G) Root hair close-up. Bar = 100 μm.
(H) Flower bud.
(I) Open flower.
(J) Mature leaf.
(K) Stem and siliques. Inset: silique detail with developing seeds.
- 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 ± sd.
- 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 determined 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 ± se of two independent experiments.
- 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 = 100 μm.
(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).
- 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), ΔNT-MORN (PIPK3228-705; [D]); K442A (E), and TripleA (F). Bars = 200 μm. 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 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 ± se of two independent experiments.
- 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 = 200 μm in (A) to (D) and 100 μm (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 ± 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.
- Figure 7.
Effects of K442A or ΔNT-MORN Expression on Root Hair Growth.
(A) to (D) The inactive PIP5K3-derived K442A protein or the truncated ΔNT-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 (ΔNT-MORN) independent lines tested. To varying degrees, all lines also exhibited regions with unaltered root hairs. Bars = 200 μm.
(A) Wild-type control.
(B) RedStar control.
(C) and (D) Root hair morphology observed with expression of K442A (C) or ΔNT-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 (antisense). 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 ± se of two independent experiments. 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.
- 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 promoter. Bars = 20 μm.
(A) Bright-field image.
(B) EYFP-PIP5K3 fluorescence.
(C) Merge of images (A) and (B).
(D) Magnified view of a 1.0-μm 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 μm) 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.
- 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 RedStar-tagged HsPLCδ1-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 = 10 μm.
(A) PIP5K3.
(B) K442A.
(C) Quantification of relative fluorescence intensities in horizontal sections 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 representative 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.
Tables
Substrate PtdIns4P/PtdIns3P Protein PtdIns3P PtdIns4P PtdIns5P Activity Ratio MBP 2 ± 0.3 4 ± 0 – – PIP5K1 5 ± 0.8 205 ± 17 – 41.0 PIP5K2 52 ± 8 1206 ± 156 – 23.0 PIP5K3 178 ± 27 1972 ± 66 – 11.0 PIP5K7 2 ± 0.3 37 ± 6 – 18.5 PIP5K8 3 ± 0.5 27 ± 3 – 9.0 PIP5K9 4 ± 0.6 142 ± 23 – 35.5 Catalytic activities were tested in vitro against different phosphatidylinositol-monophosphate substrates and indicate the rate of product formation in fmol min−1. Recombinant protein concentrations were balanced according to protein gel blot analysis. Data are the means of two independent experiments ± sd.
Supplemental Data
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