Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase.
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.
Perera
Imara Y
IY
Department of Botany, North Carolina State University, Raleigh, North Carolina 27695, USA. imara_perera@ncsu.edu
Love
John
J
Heilmann
Ingo
I
Thompson
William F
WF
Boss
Wendy F
WF
D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D000072181:Inositol Polyphosphate 5-Phosphatases; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D030821:Plants, Genetically Modified; D014026:Nicotiana / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation; D014862:Wasp Venoms / Q000737:chemistry / Q000378:metabolism
D036341:Intercellular Signaling Peptides and Proteins; C084070:Mas7 protein, synthetic; D010455:Peptides; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010761:Phosphorus Radioisotopes; D014862:Wasp Venoms; C037500:mastoparan; D015544:Inositol 1,4,5-Trisphosphate; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases
NASA Discipline Cell Biology; NASA Discipline Plant Biology; Non-NASA Center