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-Dglucopyranoside 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 ...

Whole-Nodule Carbon Metabolites Are Not Involved in the Regulation of the Oxygen Permeability and Nitrogenase Activity in White Clover Nodules

“…1). Nodule Glc concentration was very low and near the limit of detection, as was also found by Davis and Nordin (1983). Since Gordon et al (1990) provided evidence for the presence of Suc synthase in white clover nodules, this resuIt may indicate that Suc in white clover nodules is cleaved mainly by Suc synthase, because UDP-Glc, the product resulting from this cleavage, cannot be detected by the method used in this study.…”

“…A11 samples were filtered (LS 14, Schleicher & Schuell) and diluted with water to 10 mL. For Glc determination, 0.05-mL aliquots were added to a mixture of 0.6 mL of triethanolamine-HC1 buffer (750 mM, pH 7.6) with 900 mM ATP and 13 miv NADP, 0.98 mL of water, and 0.01 mL of hexokinase (450 units Gordon et al (1986) and Minchin et al (1986); the present data on organic acids are substantially higher (more than a factor of 10) compared to those described by Davis and Nordin (1983). Different growing conditions, plant age, nutrient status, plant and/or bacteria cultivars, or methodological reasons could explain such differences.…”

Carbohydrate, Organic Acid, and Amino Acid Composition of Bacteroids and Cytosol from Soybean Nodules

“…We found this approach to be very insensitive, requiring a very large sample size (>5 g fresh weight of nodules) for analysis of acids in bacteroids. Also, the resolving power ofthese columns was poor, with several peaks containing more than one compound (4).…”

Effects of Salt Stress on Amino Acid, Organic Acid, and Carbohydrate Composition of Roots, Bacteroids, and Cytosol of Alfalfa (Medicago sativa L.)

“…As in nodules from white clover (3) or in the cytosol of B. japonicum nodules (21), sucrose was one of the most abundant sugars. Pinitol was the second major carbohydrate, but its concentration was much lower than that of sucrose: 14% in cytosol and 13% in roots.…”