Regulation of β-Glucan Synthetase Activity by Auxin in Pea Stem Tissue

Abstract

The 2-to 4-fold rise in particle-bound 3-glucan synthetase (uridine diphosphate-glucose:,6-1 ,4-glucan glucosyltransferase) activity that can be induced by indoleacetic acid in pea stem tissue is not prevented by concentrations of actinomycin D or cycloheximide that inhibit growth and macromolecule synthesis. The rise is concluded to be a hormonally induced activation of previously existing, reversibly deactivated enzyme. The activation is not a direct allosteric effect of indoleacetic acid or sugars. It is blocked by inhibitors of energy metabolism, by 2-deoxyglucose, and by high osmolarity, but not by Ca'+ at concentrations that inhibit auxin-induced elongation and prevent promotion of sugar uptake by indoleacetic acid, and not by a, a'-dipyridyl at concentrations that inhibit formation of hydroxyproline. Regulation of the system could be due either to an ATP-dependent activating reaction affecting this enzyme, or to changes in levels of a primer or a lipid cofactor.Under conditions previously defined (37), treatment of pea stem tissue with IAA or other auxins causes a rapid, temperature-dependent rise in the level of particle-bound /8-glucan synthetase activity (UDP-glucose:/8-1 ,4-glucan glucosyltransferase) obtainable from the tissue. This report considers how this hormonal effect depends upon cellular metabolism. A somewhat similar investigation of the effect of IAA, applied in lanolin paste, on GDP-glucose-dependent glucan synthetase activity in stem tissue of whole decapitated pea seedlings was published recently (48). METHODSPlant material and glucan synthetase preparation and assay were as described previously (37). Briefly, pea stem segments 8 mm long cut from the region of maximal elongation rate were kept in a moist atmosphere at 35 C for 2.5 to 3 hr to reduce glucan synthetase activity substantially below the initial level; then a medium containing usually 30 mm sucrose was added and incubation at 35 C was continued for 1 hr. Then the tissue was treated with plus-or minus-IAA media at a specified temperature. In the case of inhibitor experiments the media also contained 16 mm K phosphate buffer, pH 6.1, and 'Supported by a grant from the National Science Foundation. when necessary to ensure establishment of the appropriate inhibition by the time of addition of IAA the sucrose pretreatment media also contained the inhibitor, as specified for the experiments reported. At the end of the treatment period the tissue was chilled on ice, washed with ice water, and used for preparation and assay of glucan synthetase (37).In Vivo Isotope Incorporation Experiments. Samples of 50 segments were incubated in 3.0 ml of labeled substrate in 16 mM K phosphate buffer, pH 6.1, on a reciprocating shaker (120 oscillations/min) at 25 C. At the conclusion of incubation the segments were washed thoroughly with ice water. For assay of incorporation from sugars into particles and cell wall the segments were ground in the same manner (37) as for glucan synthetase assay; the cell wall residue (1,OOOg) and particle pe...

Bound Auxin Formation in Growing Stems

“…Osmotic solutes or Ca2`outside the stem had no effect showing that cell expansion is not a requisite for auxin binding. 2-Deoxyglucose, which inhibits the incorporation of sugars into wall polymers (16), only had an effect on the water-soluble fraction. These results are at variance with those found by Ray (16) for auxininduced glucan synthesis which did not require protein or RNA synthesis, though glucan synthesis was assayed after only 2 hr of inhibitor treatment so the glucan synthetase enzymes relying on IAA stimulation might simply have a life in excess of 2 hr.…”

Regulation of β-Glucan Synthetase Activity by Auxin in Pea Stem Tissue

“…However, correspondence in time between stimulated elongation and onset of rise in synthetase activity suggests a close connection between them, for example, possibly stemming from onset of enhanced export of polysaccharide products into the cell wall. That the induction of synthetase activity could be merely a consequence of the occurrence of rapid elongation is ruled out by the observations (a) that a substantial effect of IAA is detectable in subapical stem segments that are capable of little or no cell enlargement in response to IAA (Table II), and (b) that IAA exerts a substantial effect on synthetase activity in the presence of concentrations of Ca2+, actinomycin D, or cycloheximide that severely inhibit elongation (17).…”

“…This dependence upon metabolism is explored in the second report of this series (17), which indicates that the phenomena involve deactivation and activation of pre-existing enzyme rather than enzyme destruction and new enzyme synthesis. In accord with this are the data that suggest that IAA causes an increase in affinity for UDP-glucose (Fig.…”

Activation of β-Glucan Synthases by Wall-Bound Purple Acid Phosphatase in Tobacco Cells

“…A more recent report on the identification of the phosphorylation sites of AtCesA7, which is a cellulose synthase subunit involved in secondary cell wall formation in Arabidopsis, points toward a role of phosphorylation events in regulating the turnover of cellulose synthase by proteolysis through a proteasomedependent pathway (Taylor, 2007). The implication of phosphorylation in the regulation of b-glucan synthases has also been proposed in pea (Pisum sativum) stems (Ray, 1973). In addition, sodium fluoride, which inhibits the action of phosphoprotein phosphatases was shown to increase by 5-fold b-glucan synthase activity from corn (Zea mays) in the presence of Ca 2+ , possibly in a calmodulindependent manner (Paliyath and Poovaiah, 1988).…”