Determination of Rhizobitoxine and Dihydrorhizobitoxine in Soybean Plants by Amino Acid Analyzer

Abstract

Rhizobitoxine (RT) (2-amino-4-(2-amino-3-hydropropoxy)-trans-but-3-enoic acid, C7H14N204=190) is a phytotoxin produced by some strains of Bradyrhizobiumjaponicum (1-3). ART-producing B. japonicum often induces chlorosis in new leaves of the host plant as a result of the synthesis of the toxin in the nodules (l, 4). Physiologically, RT is known to inhibit fi-cystathionase in the methionine biosynthetic pathway and ethylene formation (5-7). The ability to produce RT has been reported to be common among Bradyrhizobium strains originating from the U .S.A., Africa, Central America, and South America (8). Dihydrorhizobitoxine (DRT), a structural analogue of RT, is also associated with the occurrence of RT in nodules arid has been identified as O_(2_amino-3-hydroxylpropyl)homoserine (C 7 H 16 N 2 0,=192) (1, 2, 9). RT has been detected semi quantitatively by paper chromatography or bioassay. OWENS and WRIGHT (1, 2) separated RT and DRT (unknown Y) by two-dimensional paper chromatography, detecting them by spraying ninhydrin, and the amounts of RT and DRT were relatively estimated by visual comparison with asparagine standards and alanine (or purified unknown Y) standards, respectively. LA FAVRE and EAGLESHAM (8) separated RT by one-dimensional paper chromatography, and the color density of its yellow reaction product with ninhydrin on paper was classified into 3 levels. The relative amount of RT was also assayed by the degree of chlorosis of soybean plants (1,4,8) and seedlings of various plants (4). We have not been able to find any reference on the accurate determination of RT. This report describes the quantitative measurement of the RT and DRT contents of soybean plants using an amino acid analyzer.RT and DRT were isolated and purified from whole-culture extracts of strain. of B. japonicum USDA 94 using the procedure of GIOVANELLI (6), except for the addition of ion exchange chromatography (pyridine type (10» before the re-chromatography step. Fifteen mg of RT and 22 mg of DRT were purified from 7 liters of culture.

Rhizobitoxine inhibition of hydrogenase synthesis in free-living Bradyrhizobium japonicum

“…The concentration of rhizobitoxine was measured with an amnino acid analyzer (14). The cell suspensions were centrifuged at 15,000 x g for 5 min.…”

“…The column was washed with distilled water and eluted with 5 bed volumes of 2 M NH40H. The eluate fraction was evaporated to dryness and dissolved in dilution buffer (14).…”

“…Rhizobitoxin had no effect on hydrogenase activity but inhibited derepression of hydrogenase. (14). Other reagents were special-grade products from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).…”

Bradyrhizobium elkanii rtxC Gene Is Required for Expression of Symbiotic Phenotypes in the Final Step of Rhizobitoxine Biosynthesis

“…The deduced amino acid sequence of rtxC showed 31% similarity to Pseudomonas fatty acid desaturase. From these results, we proposed that rtxC protein is involved in desaturation of dihydrorhizobitoxine, although possible polar effects of the kanamycin resistance cassette and cosmid instability may have complicated this interpretation.A large amount of dihydrorhizobitoxine, an oxidative form of rhizobitoxine, is generally coproduced with rhizobitoxine (20,26). Biochemical analysis indicates that the ability of dihydrorhizobitoxine to inhibit ACC synthase and ␤-cystathionase is approximately 99% less than that of rhizobitoxine (39).…”

“…A large amount of dihydrorhizobitoxine, an oxidative form of rhizobitoxine, is generally coproduced with rhizobitoxine (20,26). Biochemical analysis indicates that the ability of dihydrorhizobitoxine to inhibit ACC synthase and ␤-cystathionase is approximately 99% less than that of rhizobitoxine (39).…”