Enzymes of α,α-Trehalose Metabolism in Soybean Nodules

Abstract

Metabolism of trehalose, a,D-glucopyranosyl-a,D-glucopyranoside, was studied in nodules of Bradyrhizobium japonicum-Glycine max IL.I Merr. cv Beeson 80 symbiosis. The nodule extract was divided into three fractions: bacteroid soluble protein, bacteroid fragments, and cytosol. The bacteroid soluble protein and cytosol fractions were gel-filtered. The key biosynthetic enzyme, trehalose-6-phosphate synthetase, was consistently found only in the bacteroids. Trehalose-6-phosphate phosphatase activity was present both in the bacteroid soluble protein and cytosol fractions. Trehalase, the most abundant catabolic enzyme was present in all three fractions and showed two pH optima: pH 3.8 and 6.6. Two other degradative enzymes, phosphotrehalase, acting on trehalose-6-phosphate forming glucose and glucose-6-phosphate, and trehalose phosphorylase, forming glucose and 0-glucose-l-phosphate, were also detected in the bacteroid soluble protein and cytosol fractions. Trehalase was present in large excess over trehalose-6-phosphate synthetase. Trehalose accumulation in the nodules would appear to be predicated on spatial separation of trehalose and trehalase.Trehalose, a-D-glucopyranosyl-a-D-glucopyranoside, is one of the major carbohydrates synthesized by cultured Bradyrhizobium japonicum, and every species of Rhizobium examined so far shows trehalose accumulation (26). This disaccharide has also been reported in yeast (1,5), in fungi (12, 22), as well as in bacteria (3), mainly in actinomycetes (15). In soybean plants trehalose appears to be restricted to the nodule tissue (23). Its presence elsewhere in higher plants has not been conclusively established (7). Previous work with soybean nodules suggests that trehalose is synthesized by the microsymbiont (20).The pathway for trehalose synthesis was first elucidated in Saccharomyces cerevisiae (5) (Fig. 1). The biosynthetic enzymes have also been reported in Lilium longiflorum pollen (8).There appears to be more than one way of catabolizing trehalose (Fig. 1). Phosphotrehalase has been reported in Bacillus popilliae (3) and trehalose phosphorylase in Euglena gracilis (17). Trehalase, the most widely occurring catabolic enzyme has also been found in higher plants (6,9,27 Nodule Fractionation. After 5 to 6 weeks the plants were harvested, and subsequently all operations were conducted at 0 to 2°C. Five g of nodules were ground in a mortar with a pestle in 10 ml (added in 2 ml aliquots) of grinding medium, 0.1 M sodium phosphate buffer (pH 7.5) containing 0.15 M mannitol, 2 mm DTE, and 1 mm EDTA. The homogenate was filtered through two Miracloth discs in a 50 ml syringe into a 15 ml centrifuge tube.The Miracloth filtrate was then centrifuged at 5OOg for 10 min. The resultant starch pellet was discarded and the supernatant fluid recentrifuged at 6,000g for 10 min to obtain the bacteroid pellet. The supernatant fluid was centrifuged at 48,000g for 15 min and the supernatant, cytosol fraction, was saved for gel filtration.The bacteroid pellet was resuspended in 8 ml of grindin...

Functional Role of Bradyrhizobium japonicum Trehalose Biosynthesis and Metabolism Genes during Physiological Stress and Nodulation

“…Therefore, it would seem logical that trehalose can be used to support growth of this bacterium. However, earlier studies have shown that B. japonicum cannot utilize trehalose as a sole source of carbon for growth (34), despite the fact that B. japonicum bacteroids have also been reported to have trehalase, an enzyme catalyzing degradation of trehalose into two molecules of glucose (27,28,33). Interestingly, strain USDA 110 contains malQ (bll6765) and aglA (blr0901), and these genes, as well as the trehalose biosynthetic genes, were highly induced by desiccation stress (6).…”

Three Enzymes for Trehalose Synthesis in Bradyrhizobium Cultured Bacteria and in Bacteroids from Soybean Nodules

“…TPS functions with various nucleotide phosphate derivatives of glucose, and polyanions such as heparin are strong activators of TPS with some NDP-glucose substrates (11,20). The presence of TPS has previously been reported in studies of Bradyrhizobium japonicum and B. elkanii, the organisms of interest here, although the levels of activity were low (22).…”

“…Nodules were weighed and ground in a cold mortar with 100 mM phosphate buffer (pH 7.5) containing 150 mM mannitol, 2 mM dithiothreitol, and 1 mM EDTA (22). After filtration through four layers of cheesecloth, the homogenate was centrifuged at 500 ϫ g for 10 min to remove debris.…”

“…The assay of trehalose-6-phosphate synthase was performed to detect glucose-6-phosphate-dependent formation of UDP from UDP glucose (3,22). Substrates were added to gel-filtered protein to maintain a high protein concentration; heparin was included at a final concentration of 40 g per ml.…”

The thuEFGKAB Operon of Rhizobia and Agrobacterium tumefaciens Codes for Transport of Trehalose, Maltitol, and Isomers of Sucrose and Their Assimilation through the Formation of Their 3-Keto Derivatives

“…Because rhizobia do not possess a complete PTS for carbohydrate uptake (15), the two latter pathways described are unlikely to exist in rhizobia. Trehalase has been detected in Glycine max nodules (5,16), but except for ␣-glucosidases, which work poorly on trehalose (17), enzymes involved in trehalose catabolism have not been unequivocally identified in free-living rhizobia (18). Previously, we identified and characterized the trehalose-inducible thu operon, thuEFGKAB, involved in the transport and assimilation of trehalose in Sinorhizobium meliloti and demonstrated that the genes thuEFGK of this operon code for an ATP-binding cassette for transport of trehalose and that disruption of thuAB sequences results in accumulation of trehalose in the cells, implying their role in trehalose utilization.…”