An Arabidopsis inositol phospholipid kinase strongly expressed in procambial cells: Synthesis of Ptdlns(4,5)P-2 and Ptdlns(3,4,5)P-3 in insect cells by 5-phosphorylation of precursors

We have cloned a phosphatidylinositol-4-phosphate 5-kinase (PIP5K) cDNA (At P5K1) from Arabidopsis thaliana. By the application of cell permeabilizatio n and short-term nonequilibrium labelling we show that expression of AtP5K1 in Baculovirus-infected insect (Spodoptera frugiperda) cells directs synth esis of PtdIns(4,5)P-2 and PtdIns(3,4,5)P-3. The same phosphoinositides wer e produced by isolated whole-cell membrane fractions of AtP5K1-expressing i nsect cells. Their synthesis was not affected by adding defined precursor l ipids, that is PtdIns(3)P, PtdIns(4)P, PtdIns(3,4)P-2, or PtdIns(4,5)P-2, i n excess, indicating that substrates for the plant enzyme were not limiting in vivo. Enzymatic dissection of lipid headgroups revealed that AtP5K1-dir ected synthesis of Ptdins(4,5)P-2 and PtdIns(3,4,5)P-3 proceeds via 5-phosp horylation of precursors. Analysis of promoter-reporter gene (beta -glucuro nidase) fusions in transgenic plants revealed that expression of the AtP5K1 gene is strongest in vascular tissues of leaves, flowers, and roots, namel y in cells of the lateral meristem, that is the procambium. Single-cell sam pling of sap from flower stem meristem tissue and neighbouring phloem cells , when coupled to reverse transcriptase - polymerase chain reaction, confir med 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.