Increasing phosphatidylinositol (4,5) bisphosphate biosynthesis affects plant nuclear lipids and nuclear functions

Abstract

In order to characterize the effects of increasing phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P(2)) on nuclear function, we expressed the human phosphatidylinositol (4)-phosphate 5-kinase (HsPIP5K) 1α in Nicotiana tabacum (NT) cells. The HsPIP5K-expressing (HK) cells had altered nuclear lipids and nuclear functions. HK cell nuclei had 2-fold increased PIP5K activity and increased steady state PtdIns(4,5)P(2). HK nuclear lipid classes showed significant changes compared to NT (wild type) nuclear lipid classes including increased phosphatidylserine (PtdSer) and phosphatidylcholine (PtdCho) and decreased lysolipids. Lipids isolated from protoplast plasma membranes (PM) were also analyzed and compared with nuclear lipids. The lipid profiles revealed similarities and differences in the plasma membrane and nuclei from the NT and transgenic HK cell lines. A notable characteristic of nuclear lipids from both cell types is that PtdIns accounts for a higher mol% of total lipids compared to that of the protoplast PM lipids. The lipid molecular species composition of each lipid class was also analyzed for nuclei and protoplast PM samples. To determine whether expression of HsPIP5K1α affected plant nuclear functions, we compared DNA replication, histone 3 lysine 9 acetylation (H3K9ac) and phosphorylation of the retinoblastoma protein (pRb) in NT and HK cells. The HK cells had a measurable decrease in DNA replication, histone H3K9 acetylation and pRB phosphorylation.

Figure 1

Nuclei isolated from NT and HK cells are intact and have similar size. NT (A) and HK (B) cells. HK cells are slightly smaller. NT (C) and HK (D) nuclei after isolation. Note Nucleolus denoted by arrows.

Figure 2

DGDG and MGDG from NT and HK nuclei and protoplast PM samples. NT nuclei (white), NT protoplast PM (white dotted), HK nuclei (grey) and HK protoplast PM (grey striped). Nuclei data are represented by the average mol % ± standard error (SE). NT nuclei data is the average of 10 samples from 7 different biological replicates and HK nuclei data is the average of 12 samples from 8 different biological replicates. Protoplast PM data are the average of 5 lipid extractions from 3 different biological replicates. Significant changes in the MGDG and DGDG were not found when comparing NT to HK nuclear lipid samples, but a significant increase was seen in the HK protoplast PM MGDG compared to the NT protoplast PM MGDG. In addition, there is a 1.4 fold increase in MGDG on a mol % basis in the PM of both types of protoplasts compared to the respective nuclei.

Figure 3

HK nuclei have increased steady state PtdIns(4,5)P₂. Lipid headgroup analysis of steady state PtdIns(4,5)P₂ was measured from NT (white) and HK (grey) nuclei. Measurements are the average of 3 independent nuclei isolations ± Stdev. ND = not detected. At least 0.5 mg of nuclear protein was used per sample.

Figure 4

HK nuclei have increased endogenous PIP5K activity. NT (white) and HK (grey) nuclei were assayed for endogenous lipid kinase activity without PtdIns4P added (−) and added PtdIns4P (+). [³²P]PtdInsP₂ (A, D), [³²P]PtdInsP (B, E) and [³²P]PtdOH (C, F) are reported as pmol.mg⁻¹.min⁻¹. Measurements are the average of 3 independent experiments ± SE.

Figure 5

Lipid classes from NT and HK nuclei and protoplast PM show significant differences between cell types. A. Comparison of lipid classes from NT (white) and HK (grey) nuclei. B. Comparison of lipid classes from NT (white dotted) and HK (grey striped) protoplast PM. C. Comparison of lipid classes from NT nuclei (white) and NT protoplast PM (white dotted). D. Comparison of lipid classes from HK nuclei (grey) and HK protoplast PM (grey striped). * indicates a changed lipid classes at a p-value of 0.05. NT nuclei data are the average of 10 lipid extractions from 7 independent biological replicates. HK nuclei data are the average of 12 samples from 8 independent biological replicates. NT and HK protoplast PM data are the average of 5 lipid extractions from 3 independent biological replicates. All data are the average mol % ± SE.

Figure 6

BrdU incorporation is decreased in HK cells compared with NT cells. A) Example of an immunoblot detecting BrdU incorporation in DNA isolated from NT and HK cells after BrdU treatment. Three different DNA concentrations are shown. B) A comparison of BrdU incorporation between NT and HK cells, normalized to total signal intensity (L=low, M=medium and H=high DNA, e.g. 50, 75 and 100 ng of DNA). Values are the average of more than three separate experiments ± SE.

Figure 7

Histone H3 lysine 9 acetylation is decreased in HK cells compared with NT cells. A) Example of an immunoblot with H3K9ac-specific antibodies and H3-specific antibodies with NT and HK protein samples. B) Graph of the average ratio of H3K9ac/H3 ratio from HK cells compared to the NT H3K9ac/H3 ratio which was normalized to 100%.

Figure 8

Phosphorylation of retinoblastoma protein (pRb) is decreased in HK cells. Immunoblot is shown using pRb phosphoSer807/811 specific antibodies of two biological replicates of NT and HK cell samples. Arrow indicates the migration of pRb at ~100kDa.