Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase

Abstract

To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.

Figure 1

Expression of the human InsP 5-ptase gene in the transgenic tobacco cells. A, Schematic of the binary plasmid containing the InsP 5-ptase construct used to transform tobacco cells. 35S, Cauliflower mosaic virus 35S promoter; 6 His, His tag; human InsP 5-ptase I, coding region of the human type I InsP 5-ptase gene; rbcS 3′, 3′-untranslated region of the pea (Pisum sativum) rbcS E-9 gene; X, XbaI restriction enzyme sites; Km^R, kanamycin resistance cassette; black arrows, T-DNA border sequences. B, RNA gel-blot analysis. RNA was isolated from transgenic and wild-type cell lines harvested on d 4 of the culture cycle. Total RNA (5 μg lane⁻¹) was electrophoresed on a formaldehyde agarose gel and transferred to a nylon membrane. The blot was probed with ³²P-labeled InsP 5-ptase cDNA probe (upper) and stained with methylene blue to visualize RNA loading (lower). The arrow points to an approximately 1.7-kb transcript corresponding to InsP 5-ptase. WT, Wild type; C-3 and C-5, vector controls; I2-2, I2-8, and I2-11, transgenic lines transformed with InsP 5-ptase. C, Western blot of cell lysates. Cell lysates from transformed and wild-type tobacco cells were separated by SDS-PAGE electrophoresis (20 μg protein lane⁻¹), transferred to polyvi- nylidene difluoride (PVDF) membrane, and immunostained using antisera raised against the His tag. The antiserum recognizes a protein of approximately 45 kD that is detectable only in cells transformed with the InsP 5-ptase construct. The size of the protein expressed in tobacco cells is comparable with the purified recombinant InsP 5-ptase protein produced in bacteria pQE31 (<0.5 μg lane⁻¹). The lower panel shows the same blot stained with Amido black to visualize total protein.

Figure 2

Cell growth and morphology of wild-type and transgenic tobacco cells. A, Cell samples from 5-mL cultures were harvested daily over the culture cycle and the fresh weight monitored. Three replicate cultures of each sample were harvested at each time point. A representative growth curve (drawn through the average values for each time point) from one experiment is shown in the figure. The bars show the range in fresh weight values obtained from the replicate cultures at each time point. The experiment was repeated twice with similar results. B, Fluorescence microscopy of cells from d 4 of the culture cycle. Cells were stained with 0.01% (w/v) fluorescein diacetate in acetone. The scale bar represents 25 μm.

Figure 3

Increased activity of the InsP 5-ptase protein and decreased basal InsP₃ levels in the transgenic tobacco cells. A, InsP 5-ptase activity of cell lysates. Microsomal (M) or soluble (S) protein fractions (30 μg protein assay⁻¹) from transformed and wild-type tobacco cells were incubated for 10 min at room temperature with commercial InsP₃. Relative InsP 5-ptase activity was plotted as the percentage of InsP₃ hydrolyzed by each fraction compared with the wild-type control. The data are the averages of three independent experiments assayed in duplicate. The variation between experiments is <5%. Sample I2-11 was only assayed as a total protein extract. B, InsP₃ levels of wild-type and transformed cells from d 4 of the culture cycle were measured as described in “Materials and Methods.” Data plotted are the average of four independent experiments assayed in duplicate.

Figure 4

InsP₃ changes in response to mastoparan treatment. Changes in InsP₃ levels in response to 5 μm Mas-7 (solid lines) or 5 μm Mas-17 (dotted lines) in 4-d-old wild-type cultures (white circles) and InsP 5-ptase-expressing cell line I2-8 (black diamonds) over the first few minutes after stimulation. The data plotted are the average of four independent experiments assayed in duplicate and the error bars show the range.

Figure 5

Altered PI metabolism in transgenic tobacco cells. A, PtdInsP₂ content of wild-type and transformed cells from d 4 of the culture cycle were measured as described in “Materials and Methods.” Data plotted are the average of two independent experiments assayed in duplicate. B, Specific activity of PtdInsP 5 kinase from microsomes and plasma membranes and specific activity of PLC from plasma membranes at d 4 of the culture cycle. The specific activity of PtdInsP 5 kinase (PIPK) from microsomes (M) and plasma membrane-enriched fractions (PM) and the PLC activity from plasma membrane fractions of the transgenic tobacco cell lines was plotted as percentage of the wild type (set at 100%). Wild-type PtdInsP 5 kinase activity was typically 12 and 180 pmol min⁻¹ mg⁻¹ for microsomes and plasma membranes, respectively. PLC activity of plasma membranes from wild type was 1 to 2 nmol min⁻¹ mg⁻¹ membrane protein. The samples were assayed in duplicate and the values plotted are the average of three independent experiments. The error bars show the range. C, Incorporation of ³²Pi into PtdInsP₂ in wild-type (open squares) and transgenic (closed diamonds) tobacco cells over a 10-min labeling period. Equivalent cell samples were removed at the specific time points and the lipids were extracted, separated by thin-layer chromatography (TLC), and quantified as described in “Materials and Methods.” Data plotted are the average of duplicates from a representative experiment. Similar results were obtained in two independent experiments.