An Arabidopsis inositol phospholipid kinase strongly expressed in procambial cells: synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in insect cells by 5-phosphorylation of precursors.
We have cloned a phosphatidylinositol-4-phosphate 5-kinase (PIP5K) cDNA (AtP5K1) from Arabidopsis thaliana. By the application of cell permeabilization and short-term nonequilibrium labelling we show that expression of AtP5K1 in Baculovirus-infected insect (Spodoptera frugiperda) cells directs synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3. The same phosphoinositides were produced by isolated whole-cell membrane fractions of AtP5K1-expressing insect cells. Their synthesis was not affected by adding defined precursor lipids, that is PtdIns(3)P, PtdIns(4)P, PtdIns(3,4)P2, or PtdIns(4,5)P2, in excess, indicating that substrates for the plant enzyme were not limiting in vivo. Enzymatic dissection of lipid headgroups revealed that AtP5K1-directed synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 proceeds via 5-phosphorylation of precursors. Analysis of promoter-reporter gene (beta-glucuronidase) fusions in transgenic plants revealed that expression of the AtP5K1 gene is strongest in vascular tissues of leaves, flowers, and roots, namely in cells of the lateral meristem, that is the procambium. Single-cell sampling of sap from flower stem meristem tissue and neighbouring phloem cells, when coupled to reverse transcriptase--polymerase chain reaction, confirmed preferential expression of AtP5K1 in procambial tissue. We hypothesize that AtP5K1, like animal and yeast PIP5K, may be involved in the control of cell proliferation.
Elge
S
S
Max-Planck-Insitute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Golm, Germany.
Brearley
C
C
Xia
H J
HJ
Kehr
J
J
Xue
H W
HW
Mueller-Roeber
B
B
D000818:Animals; D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D008969:Molecular Sequence Data; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000096:biosynthesis*; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D018411:Spodoptera
D017931:DNA Primers; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase