Sugar and Organic Acid Constituents in White Clover

Abstract

Major ethanol-soluble carbohydrate and organic acid constituents of white clover (Trifolium repens) have been identified by use of high-performance liquid chromatography and gas chromatography. In leaves, petioles, roots, and nodules, pinitol (3-0-methyl chiro-inositol) is the predominant sugar, with sucrose present in lower concentration. In leaves and petioles there are significant levels of a-and fl-methyl glucosides, linamarin, glucose, and fructose. In the nodules glucose is rarely present at detectable levels. The concentration of pinitol is generally greater than 25 millimolar in each tissue examined whereas the level of sucrose varies depending on the time of day. Sucrose is the major sugar significantly labeled during 1 hour administration of '4C02 and accounts for more than 99%o of al the radioactivity detected in the nodules at early times. Between 3 and 7 hours after labeling, 6% of the radioactivity is found in the organic acids fraction and 5% in the basic fraction of nodules. Malonic acid does not appear to be present in unusually high concentrations in either leaves or nodules of white clover. Considerable effort has been expended to identify the major ethanol-soluble carbohydrates (hereafter referred to as sugars) and organic acid constituents of soybean, an economically important legume (5-7, 13, 14, 17, 19-22). Much less has been done with other legumes (1, 18). We have examined white clover (Trifolium repens L.) because it is a convenient test organism for studies of the fast-growing Rhizobium trifolii. It has been much easier to induce expression of nitrogenase ex planta with the slow-growing brady rhizobia (16) than with the fast growers, and the fast growers may have different requirements for symbiotic induction that can be met only by particular species of legumes. We suspected that clover might contain a specific carbon substrate needed for expression of nitrogenase activity by R. trifolii. Phillips and Smith (17) reported that soybean (Glycine max [L.] Merr.) and other legumes contained high levels of a methyl inositol. Streeter (19, 20) showed that this compound was D-pinitol (3-0-methyl chiro-inositol) and studied its seasonal appearance and disappearance in soybeans. Other sugars present in significant quantities included myo-inositol, sucrose, fructose, and glucose. Smith and Phillips (18) reported the presence of 1-0-methyl f-D-glucopyranoside in leaves and petioles of white clover. Linamarin, a cyanogenic glucoside, is also present in these tissues (12). Stumpf and Burris examined the organic acid constituents of soybean and found that malonic acid was a major constituent (21). Lower levels of the Krebs cycle acids also were present and readily detected in leaves and nodules. Malonic acid was rapidly labeled by feeding "CO2 to root nodules (22). In agreement with Phillips and Smith (17), we have found the ' Supported by the Kansas Agricultural Experiment Station, contribution 83-29-j. methyl inositol, pinitol, to be present at high concentrations in white clover. We also found f8-methyl glucose (18) and linamarin (12) in leaves and petioles. We have not found significantly higher levels of malonic acid than other organic acids in white clover nodules or leaves. MATERIALS AND METHODS Separation of Neutral Sugars. Separation and identification of aldoses and alditols by HPLC on Dionex DAx8-11 with borate buffer was previously described by Barr and Nordin (2). Replacing the copper-bicinchoninate reagent with periodate allowed detection of all sugars containing vicinal hydroxyl groups. This method has been described in detail (15). The effluent from the postcolumn reactor at 100°C was monitored with a Hitachi model 100-40 variable wavelength HPLC detector at either 270 nm with an alkaline reagent (pH 8.6) for sucrose levels of 50 to 500 nmol or at 260 nm with an acid reagent (pH 5) for sucrose at 5 to 50 nmol. Sensitivity could be increased at least 100-fold by decreasing the reagent concentration and increasing the sensitivity of the detector and recorder (4). Mobilities of sugars are expressed as a percentage of that of glucose which elutes at 87 min in this system. Some RG1C values are: a-methyl-glucoside = 11; f8-methyl-glucoside = 12; linamarin = 12; sucrose = 13; pinitol = 30; and fructose = 44. For sugar analysis, an aliquot of ethanolic extract was brought to dryness under a stream of N2 at room temperature or 50°C and redissolved in borate buffer (0.5 M, pH 8.6). Very small samples (<10 1A) in ethanol or water could be injected directly but, when injected in water, the peaks of pinitol or myo-inositol were sometimes split, suggesting that formation of stable borate complexes is a relatively slow process. Ethanol interferes with sucrose determination. Analysis of Radioactive Samples. For radioactive samples, the outlet line from the detector was shortened to give a 0.5-min delay. Samples (-0.7 ml, 1 min) were collected using a LKB 17000 Minirac into which RPI minivials were placed. Scintillation fluid (1.3 ml) consisting of 0.4% 2,5-diphenyloxazole in 2 parts toluene and 1 part Triton X-100 was then added. A turbid emulsion was obtained, but absolute counting efficiency was 77% for the acid reagent, and 70% for the alkaline reagent. Further Characterization of Sugars. Demethylation of authentic pinitol and of a nodule sample was done by treatment with 48% HBr at 4°C for 4 d (8). Under these conditions, demethylation is not complete but other sugars are not destroyed. For acid hydrol-ysis, 0.5 ml ethanolic extract from the respired nodule fraction (see Table V) was dried under N2, redissolved in 0.5 ml 2 N TFA, and heated 2 h at 100°C. It was then dried in vacuo at 4°C and reconstituted in buffer for analysis. GC was done using a Hewlett-Packard HP 5880 with a column (2 mm x 2 m) of 3% ECNSS-M on Gas Chrom Q, according to the method of Conrad et al. (3), but using 95% ethanol as the sample solvent. Preliminary analyses were done with temperature programming. Quantitation studies were done isothermally at 190°C or 200°C. Variation between integrated peak areas for a mixture of mannitol, galactitol, glucitol, and myo-inositol was only 1051