Galactose Inhibition of Auxin-Induced Growth of Mono- and Dicotyledonous Plants

Abstract

MATERIALS AND METHODS Galactose inhibited auxin-induced cell elongation of oat coleoptiles but not that of azuki bean stems. Galactose decreased the level of UDP-glucose in oat coleoptiles but not in azuki bean hypocotyls. Glucose-l-phosphate uridyltransferase activity (EC 2.7.7.9), in a crude extract from oat co-leoptiles, was competitively inhibited by galactose-l-phosphate, but that enzyme from azuki bean was not. A correlation was found between inhibition of growth by galactose and inhibition of glucose-l-phosphate ur-idyltransferase activity by galactose-l-phosphate using oat, wheat, maize, barley, azuki bean, pea, mung bean, and cucumber plants. Thus, it is concluded that galactose is converted into galactose-l-phosphate, which interferes with UDP-glucose formation as an analog of glucose-l-phosphate. Auxin-induced cell elongation of oat coleoptile segments has been reported to be inhibited by exogenously applied galactose (3, 4, 14, 20-23). In oat coleoptile segments, galactose does not affect factors involved in growth regulation such as IAA uptake, IAA metabolism, osmotic concentration of cell sap, uptake of tritiated water, respiration, auxin-induced depolymerization of xyloglucan, auxin-induced degradation of glucan, and mechanical properties of the cell wall (21). However, it strongly interferes with incorporation of labeled glucose into cell wall fractions (3, 4, 9, 21). We demonstrated that galactose inhibits auxin-induced elongation of oat coleoptile segments by inhibiting UDP-glucose formation which is a key intermediate for cell wall po-lysaccharide synthesis (9, 10). The galactose effect on UDP-glucose formation can be explained by the fact that Gal 1-P2 competitively inhibits synthesis of UDP-glucose from UTP and G 1-P by proteins of oat coleoptiles (9, 21). However, exogenously applied galactose shows very little effect on auxin-induced elongation of several dicot stem segments unlike that of oat coleoptile segments (20). There is a possibility that such a differential effect of galactose on auxin-induced growth is due to its differential effect on UDP-glucose formation and cell wall synthesis. In this study, we compared the effects of galactose on the in vitro formation of UDP-glucose in various plant species. The mechanism of the galactose inhibition was discussed. Gal 1-P, galactose 1-phosphate; G 1-P, glucose 1-phosphate; Ki, inhibition constant. Plant Materials. Oat (Avena sativa L.), wheat (Triticum aes-tivum L.), maize (Zea mays L.), and barley (Hordeum distichon L.) seeds were soaked in water for 2 h, germinated and grown on water in a plastic tray in the dark for 4 d as described previously (20). Coleoptile segments were excised from 35 to 45 mm long coleoptiles with the first leaves removed and pooled in distilled water for 2 h. Azuki bean (Vigna angularis Owhi and Ohashi), mung bean (Vigna radiata (L.) Wilczek), cucumber (Cucumis sativum L.), and pea (Pisum sativum L.) seeds were soaked in running water overnight, germinated and grown on water in a plastic tray in the dark for 6 d as described previously (20). Hypocotyl or epi-cotyl segments were excised from their upper portion and pooled in distilled water for 2 h. Growth Experiments. Organ segments (10 mm long) were floated on the test solution, (10 mM K-phosphate, pH 6.5) containing or not containing 10 AuM IAA and/or 10 mm galactose for 6 h. The segment length was measured every 2 h under dim light using a binocular microscope (x 6.3) equipped with an ocular micrometer. Determination of Sugar Phosphates and UDP-Sugars. The extraction and purification of sugar phosphates and sugar nucleotides were carried out according to the method of Carpita and Delmer (5). Five hundred segments of epicotyls stored at-20°C were extracted for 30 min with 100 ml of 80% ethanol. The segments were again extracted for 30 min with an additional 100 ml of 80% ethanol. The segments were squeezed, and the ethanol extracts were combined. To the extracts, small amounts of radioactive anionic sugars (60-80 Bq each) were added to assess the recovery during isolation as reported previously (9, 10). The combined extracts were chilled to-20°C and centrifuged at 1500g for 30 min. The supernatant solution was taken to dryness in vacuo at 40°C. The dried residue was resuspended in 10 ml of water and extracted twice with 10 ml of diethyl ether. The aqueous phase was concentrated to a small volume, and loaded onto a DEAE-Sephadex A-25 column (0.9 x 5.0 cm). The column was washed with 15 ml of distilled water, then anionic compounds were eluted with 15 ml of 0.5 M NH4HCO3. After the NH4HCO3 had been removed by drying in vacuo, the residue containing anionic compounds was resuspended in water and loaded onto a DEAE-Sephadex A-25 column (0.9 x 50.0 cm) equilibrated with 0.1 M NH4HCO3 (pH 8.0). The column was eluted with a 1 L linear gradient of 0.1 to 0.4 M NH4HCO3 (pH 8.0). Fractions of 10 ml each were collected, and A254 was measured. The total sugar content in each fraction was determined by the phenol sulfuric acid method. Fractions comprising sugar phosphates or UDP-sugars were separately combined, lyophilized, and hydrolyzed with 2 M tri-fluoroacetic acid at 121°C for 90 min. The hydrolyzed materials 1223