Intercellular Nodule Localization and Nodule Specificity of Xanthine Dehydrogenase in Soybean

Abstract

ABSTIRACIThe distribution of xanthine dehydrogenase throughout the soybean plant as well as the intercellulr localizaton of xanthine dehydrogenase within soybean nodules was determined. Polyclonal antibodies againt purified xanthine dehydrogenase were prepared and used in an enzymelinked immunosorbent assay to determine whether xanthine dehydrogenase is a nodule-specific protein. This immunological assay showed that xanthine dehydrogenase is present in far greater concentration in the nodule than in any other plant organ. Immunodiffusion tests showed that anti-soybean nodule xanthine dehydrogenase would cross-react with nodule crude extracts from the ureide producers, soybean, cowpea, and lima bean, but would not cross-react with those of the amide producers, alfalfa and lupine. A crude extract from pea nodules cross-reacted slihtly with anti-soybean xanthine dehydrogenase. Anti-soybean xanthine dehydrogenase did not cross-react with buttermilk xanthine oxidase either by enzyme-linked immunosorbent assay or by immunodiffusion test. Fresh nodule sections from the ureide-producers, soybean, cowpea, and lima bean, all stained positively for xanthine dehydrogenase. The substrate-dependent stain was inhibited by aflopurinol and was observed only in the infected nodule cells ofthese species. Nodules from the amideproducers, alfalfa and white lupine, did not stain for xanthine dehydrogenase.The ureides, allantoin and allantoic acid, are the predominant forms of nitrogen transported from nitrogen-fixing soybean nodules to other plant parts (15,23). Synthesis of ureides occurs in the plant cells of nodules via de novo purine synthesis followed by purine oxidation (19). The intracellular and intercellular localizations of the enzymes responsible for ureide synthesis has received significant attention recently (6,10,21 with a Beckman DU-7U spectrophotometer. Purification of XDH from field nodules proved to be more difficult than the procedure used for greenhouse nodules (25). All steps were performed at 4°C. The buffer used throughout the purification was 10 mm K-phosphate (pH 7.8) with I mM DTE. Field nodules (200 g) were ground in a Waring Blendor with 500 ml buffer and 50 g insoluble PVP. The extract was then centrifuged at l0,OOOg for 20 min to remove debris, bacteroids, and intact organelles. The supernatant, which is referred to as the crude extract, was brought to 30% saturation with solid (NH4)2SO4. After stirring for 20 min, the sample was centrifuged at 10,OOOg for 20 min. The supernatant was brought to 45% saturation with solid (NH4)2SO4. After stirring and centrifugation, the pellet was resuspended in 30 ml of 20% (NH4)2SO4. The resuspended pellet was then applied to a 50-ml octyl-Sepharose column equilibrated with 20% (NH4)2SO4 in buffer. A 400-ml gradient of 20 to 0% (NH4)2SO4 in buffer was applied to the column. XDH was eluted with 100 ml of the above buffer without (NH4)2SO4. Active fractions were pooled and applied to a 50-ml DEAE-Sephadex column equilibrated with buffer. A 400-ml gradient of 0 to 400 mM...

Localization of xanthine dehydrogenase in cowpea root nodules: implications for the interaction between cellular compartments during ureide biogenesis.

“…Although XDH is not nodule-specific, an elevated level of XDH is present in nodules (14,16). The higher concentration of XDH observed in the uninfected cells implies that these cells may be the site of xanthine hydroxylation for ureide biogenesis in nodules and that xanthine or a precursor to xanthine, rather than uric acid, is the intermediate transported from the infected to the uninfected cells.…”

“…We interpret the peroxisomal labeling proposed by Nguyen et al (18) to be nonspecific labeling of plastids, since their labeled organelles appear to be too large to be considered peroxisomes, whereas plastids are of this size and are known to give nonspecific labeling. Like the method of Triplett (14), that of Nguyen et al (18) probably lacks sufficient resolution for the detection and localization of XDH in uninfected cells.…”

“…The plants were grown as described (13). The polyclonal antibodies reactive with cowpea XDH used in these experiments were prepared and characterized as described (14)(15)(16). The antibodies used in this study react specifically with XDH (15).…”

Production, Characterization, and Applications of Monoclonal Antibodies Reactive with Soybean Nodule Xanthine Dehydrogenase

“…The ureides, allantoin and allantoic acid, represent the major transport form of fixed nitrogen from the nodule to the above-ground parts of soybean plants (21,25). XDH2, which catalyzes the hydroxylations of hypoxanthine and xanthine, has been purified to homogeneity from soybean nodules and its immunochemical properties have been examined (3,26,28). The enzyme is an NAD+-dependent, molybdoironflavoprotein and is present largely, if not exclusively, in the infected cells ofsoybean nodules (26,28).…”

Two Indirect Methods for Detecting Ureide Synthesis by Nodulated Legumes

“…After 10 min at room temperature, the formation of product, isoxanthopterin, was observed under long wave (365 nm) UV light by the emission of blue fluorescence. Crude nodule extracts of alfalfa, lupin, clover, pea, lima bean, cowpea, wild soybean, and soybean were prepared and assayed for XDH activity as described previously (21,23 whether the plants were inoculated with the fast-growing R. fredii or the slow-growing B. japonicum (Table I).…”