Alteration of growth and gravitropic response of maize roots by lithium.

The alkali metal lithium is known to produce a number of effects in plants and animals. Lithium salts have been an important tool in dissecting the intracellular pathways of signal transduction systems. Lithium induces an increase in diacylglycerol (DAG) in animal cell culture; the mechanism of this effect is not fully understood. Brami et al. (1993) suggest that lithium may alter the degradation of phospholipids by acting on phospholipase C or D. In the second messenger signal transduction system phosphatidylinositol-4, 5-disphosphate (PIP2) is converted into phopholipase C (PLC). Lithium can alter the second messenger signal transduction system by altering enzyme activity. Myoinositol-1-phosphatase (I-1-P) converts D-glucose-6phosphate to myo-inositol via the intermediate L-myoinositol-1-phosphate. This enzyme can hydrolyze both the Dand L-enantiomers of I-1-P. Lithium inhibits myoinositol-1-phosphatase activity in both plants and animals although plant tissue is less sensitive to the effects. In pollen of lily, 50 mM of lithium is required to produce 50% inhibition of the enzyme (Gumber et al., 1984). It has been reported that lithium salts increase inositol phosphate levels in animal tissue but that lithium has no effect on inositol phosphate levels in plants. Morse et al. (1987) found no evidence that lithium enhances the recovery of inositol phosphates in the leaf pulvina of the legume Samanea saman during the night movement of the leaves. Additionally they found that inositol is not the rate-limiting factor in the biosynthesis of phophatidylinositol in Samanea saman. Lithium salts alter ethylene biosynthesis in mung beans (Vigna radiata) (Lee and Kang, 1987). Ethylene inhibits plant growth and is produced through the conversion of methionine to s-adenylsylmethionine to aminocyclopropane carboxylic acid to ethylene. Lithium ions may alter ethylene biosynthesis by effecting the conversion of ACC to ethylene. Lithium inhibits thigmomorphogenesis in Bryonia dioica (Boyer et al., 1983) and Bidens pilosus (Desbiez et al., 1981). Rubbing the internodes of Bryonia decreased the height of the plants by inhibiting cell division and elongation. Pricking one cotyledon on a Bidens pilosus plant inhibits the growth of the hypocotyl within one hour. Lithium treatment of the plants reduced the amount of inhibition which was observed. Thus it was concluded that lithium inhibition of thigmomorphogenetic responses resulted from lithium inhibition of the effect of ethylene formed in the mechanical stimulation of the plant tissues. Plant Material: Grains of maize (Zea mays L., Pioneer 3343) were soaked overnight in running tapwater and placed between wet paper towels on opaque plastic trays. The trays were placed in a vertical position with the grains aligned along the vertical axis. The seeds were germinated at 30 C and used when the primary root is 11.5 cm in length. Measurement of Elongation: A computer-based root auxanometer system (Mulkey et al, 1982b) was used to determine the effects of lithium on elongation of intact primary roots of maize. Roots were placed into the auxanometer chamber containing half-strength Meyer's solution. Isolation of Membrane and Cytoplasmic Fractions. One-cm root tip segments (at least 3 g of roots) were ground on ice with homogenization buffer containing 50 mM MES-NaOH, pH 7.0, 5mM MgCl2, 0.5 mM DTT, 0.25 M Sucrose, 5 mM EDTA, and 0.5 mM PMSF using a mortar and pestle (1:2 w/v). The homogenate was filtered through Miracloth and centrifuged for 10 min at 7,000 g to discard nuclei and cell walls. The supernatant was collected, centrifuged for 30 min at 100,000 g for 30 min; and the supernatant was used for procedures requiring cytoplasmic fractions. The pellet was suspended in homogenization buffer at the same volume and centrifuged again at 100,000 g for 30 min. The final pellet was suspended in homogenization buffer (1 ml/5 g initial fresh weight of root), and used for procedures requiring membrane fractions. The protein content of fractions was determined by BCA Protein Assay kit. Protein Phosphorylation. In vitro protein phosphorylation assays were conducted at 30C in a total reaction volume of 100 μl containing 50 mM MES-NaOH (pH 7.0), 0.5 mM DTT, 5 mM MgCl2, 0.2 mM EGTA and 200 ug of membranous or cytoplasmic proteins. Ten μl of 1% Triton X100 was added 15 min before phosphorylation of the membrane fractions. The reaction was initiated by adding 0.037 Bq [gamma-P]-ATP. The reaction was terminated after 1 min by adding the same volume (100 μl) of electrophoresis sample buffer and by boiling the reaction mixture for 5 min. The reaction mixture of membrane protein was centrifuged at 10,000g for 5 min and the supernatant was used for analyses by SDS-PAGE. The reaction mixture of cytoplasmic protein was used without further centrifugation. Results: Figure 1 illustrates the effect of 1 mM lithium chloride on response of roots to 0.1 nM auxin. The solid line indicates that 1 mM lithium is added at 60