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

Abstract

In studies of hormonal regulation of cell wall polysaccharide synthesis, auxin-treated tissue yields greater activity of UDPglucose: /3-1 ,4-glucan glucosyltransferase (",3-glucan synthetase") than untreated controls (1,6,15,21,27). This effect may be part of the mechanism by which wall synthesis is stimulated by auxin. The present work investigates this effect as observed in isolated pea stem segments. METHODSSegments 8 mm long beginning 3 mm below the top of the apical hook were cut from the third internode of 7-day-old I Supported by a grant from the National Science Foundation.Alaska pea seedlings grown at 25 C in the dark with occasional exposure to red light. During experimental manipulation segments were handled under red light (40-w fluorescent lamp masked with red cellophane to exclude wavelengths below about 600 nm), and otherwise kept in the dark. Segments were kept in empty Petri dishes resting on ice until all those required for the experiment had been cut; the dishes were then moved to the required temperature conditions, this being regarded as zero time in the experiment. For reasons explained in "Results" the routine procedure was first to keep the segments for 2 to 3 hr at 35 C, covered with a circle (two thicknesses) of Whatman No. 1 paper moistened with distilled water, inside a closed Petri dish containing no standing liquid. The filter paper was removed and a treatment solution was added, routinely 30 or 50 mm sucrose without or with 17 bLM indoleacetic acid, and the dish was placed under the temperature conditions required in the experiment. Each sample consisted usually of 50 segments, duplicate samples normally being run and assayed for each treatment.The distribution in the pea shoot of glucan synthetase activity and of the auxin effect thereon (Table II and Fig. 1) was obtained by cutting the third internode into segments as illustrated by the diagram in Figure 1, using cutting devices with spaced razor blades, after first removing the apex (segment I) by cutting just below the point of attachment of the stipules at the third node. Four lots of 100 segments of each type were prepared, the fresh weight of each was determined, and one lot of each type ("initial" sample) was immediately used to make a glucan synthetase particle preparation as described below. The remaining three lots of each type were held for 3 hr in moist air at 35 C, at which time the second lot of each type was chilled and used for synthetase particle preparation, while the third and fourth lots were incubated for 1.5 hr at 35 C in 30 mm sucrose without or with 17 /-M IAA and then chilled, blotted, weighed, and homogenized. From the fresh weight data the auxin effect on cell enlargement was obtained as given in Table II

The Site of Cellulose Synthesis

“…Finally, it should be pointed out that studies of effects of hormones and antibiotics on levels of particulate glucan synthetase activity (14,22) have generally assumed that the changes observed result from altered biosynthesis or metabolic turnover of essential components of the active enzyme comPlant Physiol. Vol.…”

“…The possibility, however, that this enzyme may contribute to the formation of other noncellulosic products (e.g., xyloglucan) while located in endocytoplasmic membrane should not be ruled out. For example, membrane fractions from pea (14,21) and maize (3) form soluble, noncellodextrin polysaccharides from glucose in vivo. Moreover, the membrane fractions containing such newly-synthesized polysaccharides co-sediment in sucrose gradients with either Golgi (14,21) or, depending upon stage of cell growth, Golgi plus ER vesicles (21).…”

“…Because very little (<5%) of the synthetase activity recovered in these fractions was found to be associated with plasma membrane under any of the conditions used in this or previous (20,21) studies, it is referred to here as intracellular synthetase. It has been demonstrated (14,20,21) Prolonged hydrolysis of the alkali-insoluble glucan formed in these reactions using high concentration of purified /-1,4-endoglucanase (5 mg/ml of Streptomyces cellulase for 5 days at 50 C) produced only glucose and cellobiose (no laminaribiose) as the major products, plus minor components chromatographing with cellotriose and cellotetraose. Briefer incubations (1-2 days) produced these same products plus origin material which was presumably undigested cellulose.…”

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

“…Plant 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.…”

“…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.…”