Karl G. Wagner

Evidence of phosphorylated phosphatidylinositols in the growth cycle of suspension cultured plant cells

Suspension cultured cells of Catharanthus roseus rapidly consume the inorganic phosphate of the medium and incorporate about 25% of it into their phospholipids. By three different methods of analysis it was shown that from these plant cells phosphorylated phosphatidylinositol species can be extracted; the mono- and diphosphorylated species were detected in amounts of about 7 and 1%, respectively, of the phosphatidylinositol fraction. Autoradiography of 32P-labeled phospholipids showed that especially the amount of diphosphorylated species varies with the growth cycle of the suspension culture indicating that also in the higher plant cell this phospholipid turnover may play a role in the regulation of proliferation.

Evidence of phosphatidylinositol and diacylglycerol kinases in suspension cultured plant cells

Abstract Suspension cultured cells of Catharanthus roseus and Nicotiana tabacum, after two cycles of freezing and thawing, incorporated labeled phosphate from exogenous [γ-32P]ATP into their phospholipid fraction. Quantitative thin layer chromatography (TLC) revealed strongly labeled phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP) and phosphatidic acid (PA), and less incorporation into phosphatidylinositol diphosphate (PIP2). Neomycin and spermine affected the amount of phosphorylation into the different components in a similar way to that described for animal cells.

Inositol phosphates in the growth cycle of suspension cultured plant cells

Abstract Suspension cultured Catharanthus roseus and Nicotiana tabacum cells were labeled with tritiated myo-inositol and the inositol phosphates analyzed by chromatography on Dowex (1 × 8) and by HPLC. By comparison with authentic reference compounds, it was shown that the cells of both species contained inositol 1-monophosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate in a ratio of about 40 : 10 : 1 as well as large of glycerophospho-inositol, and small amounts of inositol 4-monophosphate. The low level of inositol 1,4,5-trisphosphate reflects the low content of its phospholipid precursor (PIP2) found in these cells. Further tritium labeled compounds with Rf-values of inositol 2-monophosphate, 2,4-bisphosphate and the mono- and bisphosphate of glycero-phosphoinositol were detected. The amounts and ratio of the different inositol phosphates depended on the stage of the growth cycle with the highest levels corresponding to the cell division phase; a role of the phosphatidylinositol cycle in the proliferation control of plant cells is discussed.

ENZYMATIC ACTIVITIES OF THE PHOSPHATIDYLINOSITOL CYCLE DURING GROWTH OF SUSPENSION CULTURED PLANT CELLS

Abstract Determination of individual phospholipids during the growth of a Catharanthus roseus suspension culture showed a very strong transient increase in the PI and PA content during the cell division phase. This indicates a high turnover of these phospholipids, most likely promoted via the phosphatidylinositol cycle through phosphorylation of the inositol head group. Phospholipid phosphorylation by exogenous ATP was followed with cells harvested at different points of the growth cycle. The phospholipid kinases showed peak activities in the cell division phase, whereas in the cell elongation phase lower but significant activities were observed. These results document that the phosphatidylinositol cycle is operating in cultured plant cells and obviously plays a role in growth regulation.

Plasma membrane preparations from suspension cultured plant cells contain the enzymes for the recycling of phosphatidic acid and diacylglycerol

Abstract The subcellular distribution of phosphatidylinositol(PI) kinase (EC 2.7.1.67), PI-specific phospholipase C (EC 3.1.4.3) and D, diacylglycerol (DG) kinase (EC 2.7.1.107), CTP:phosphatidic acid (PA) cytidyltransferase (EC 2.7.7.41) and PI-synthase (EC 2.7.8.11) in membrane fractions from suspension cultured Catharanthus roseus cells was investigated. Whereas activities of phospholipase C and D could be detected both in the cytosol and membrane fractions, all the other enzymes were bound to membranes. PI-kinase, membrane-bound phospholipase C and D, DG-kinase and PI-synthase had their highest specific activities in the plasma membrane enriched fraction, whereas cytidyltransferase had comparable activities both in the plasma membrane fraction and in the fractions derived from internal membranes. These data suggest that the plant plasma membrane might be autonomous with respect of the recycling of lipid cleavage products obtained by the action of phospholipase C and D.

Rapid changes in the enzyme activities and metabolites of the phosphatidylinositol-cycle upon induction by growth substances of auxin-starved suspension cultured Catharanthus roseus cells

Abstract Suspension cultured Catharanthus roseus cells grown with 2,4-dichlorophenoxyacetic acid (2,4-D) were starved for this growth substance for 3 days, thereafter cell proliferation was induced by addition of 2,4-D, kinetin, or α-naphthaleneacetic acid (α-NAA) and kinetin. In one set of experiments changes of 3H-inositol-labeled phosphoinositides and inositol phosphates were determined in a time interval of 15 min after induction. In a second set of experiments the activities of phosphatidylinositol(PI)-kinase and diacylglycerol (DG)-kinase were followed by the use of [γ-32P]ATP. Induction by 2,4-D revealed a very fast (maximum 1 min after addition of the growth substance) increase in Ins1,4P2, decrease in PIP and increase in PI-kinase activity. Addition of α-NAA and kinetin resulted in a very fast decrease of PI and PIP (1 min after induction) and a concomitant decrease in the activities of PI-kinase and DG-kinase, whereas slightly slower (2 min after induction) but very strong increases in Ins1P1 and Ins1,4P2 were observed. Although obtained with an artificial in vitro system, these results demonstrate that the activities of the PI-cycle are responsive towards phytohormones.

Phosphatidylinositol specific isoenzymes of phospholipase D from Catharanthus roseus. Purification and characterization

Abstract Microsomal membranes from suspension cultured Catharanthus roseus cells were found to contain phospholipase D activity (EC 3.1.4.4) towards phosphatidylinositol (PI). After extraction with buffer containing Triton X-100, two isoenzymes with apparent molecular weights of 50 000 and 125 000 were partially purified. The enzymes accepted only PI as substrate and transphosphatidylation activity could not be detected. Whereas several phospholipids were slightly inhibitory, phosphatidyl glycerol had stimulatory properties. The enzymes required divalent cations for activity, Ca 2+ and Mg 2+ were equally effective. In the presence of deoxycholate and Ca 2+ , K m values for PI were found to be in the range 20–40 μM. Although the smaller protein had a slightly higher temperature stability and a slightly lower p I value, both isoenzymes revealed equal enzymic properties. This is the first report on a plant PI-specific phospholipase D.

Characterization of Phosphate Uptake by Suspension Cultured Catharanthus roseus Cells

Summary The mechanism of uptake of inorganic phosphate was studied with suspension cultured Catharanthus roseus cells. The kinetics showed both a low and a high affinity phase with Km values in the IlM and mM range, respectively. Experiments with different inhibitors suggested that the high affinity phosphate transport is driven by the proton motive force at the plasma membrane and probably includes a protonxad phosphate symport. The low affinity transport revealed properties different from those of the high affinxad ity transport; its relation to phosphate transport at the tonoplast membrane is discussed. The high affinxad ity phase of Pi uptake, studied with cells from different phases of the growth cycle, revealed little changes in the Km values, which were between 2 to 51lM, whereas the Vmax values strongly depended on the growth stage with values up to 70 nmol min- 1 g-l FW.

The use of subunit exchange chromatography for the group specific fractionation of histones

Starting from the histone mixture obtained from calf thymus, the arginine rich fraction ARE+) was coupled to organomercurial agarose via a mercaptide bond to one of its cysteines. ARE-agarose proved to be useful for a large scale affinity chromatographic separation of whole histone. In 1M NaCl, pH 4.5, highly pure histone fractions could be eluted with an urea gradient revealing increasing affinity towards ARE in the order: KAP < KAS < LAK < (ARE)n(GRK)n < GRK < ARE.

Effect of cytokinins on the phospholipid phosphorylation of the suspension cultured Catharanthus roseus cells

Abstract Previous work has shown that the components (phosphorylated phosphatinositols) and enzyme activities (phosphatidylinositol kinase and diacylglycerol kinase) of the phosphatidylinositol (PI) cycle are present in suspension cultured Catharanthus roseus cells. The phospholipid kinase activities can be determined in situ by phosphorylation with labeled exogenous ATP. Incorporation of 32P into phosphorylated PI and phosphatidic acid, the products of PI kinase and diacylglycerol kinase, is reduced in the presence of low cytokinin concentrations; the concentrations for 50% inhibition are in the range 1 μM. The molecular targets of this phytohormone action are discussed.