Abstract
Adenylates (ATP, ADP, and AMP) may play a central role in the regulation of the O2-limited C and N metabolism of soybean nodules. To be able to interpret measurements of adenylate levels in whole nodules and to appreciate the significance of observed changes in adenylates associated with changes in O2-limited metabolism, methods were developed for measuring in vivo levels of adenylate pools in the cortex, plant central zone, and bacteroid fractions of soybean (Glycine max L. Merr cv Maple Arrow x Bradyrhizobium japonicum strain USDA 16) nodules. Intact nodulated roots were either frozen in situ by flushing with prechilled Freon-113(-156[deg]C) or by rapidly (<1 s) uprooting plants and plunging them into liquid N2. The adenylate energy charge (AEC = [ATP + 0.5 x ADP]/[ATP + ADP + AMP]) of whole-nodule tissue (0.65 [plus or minus] 0.01, n = 4) was low compared to that of subtending roots (0.80 [plus or minus] 0.03, n = 4), a finding indicative of hypoxic metabolism in nodules. The cortex and central zone tissues were dissected apart in lyophilized nodules, and AEC values were 0.84 [plus or minus] 0.04 and 0.61 [plus or minus] 0.03, respectively. Although the total adenylate pool in the lyophilized nodules was only 41% of that measured in hydrated tissues, the AEC values were similar, and the lyophilized nodules were assumed to provide useful material for assessing adenylate distribution. The nodule cortex contained 4.4% of whole-nodule adenylates, with 95.6% being located in the central zone. Aqueous fractionation of bacteroids from the plant fraction of whole nodules and the use of marker enzymes or compounds to correct for recovery of bacteroids and cross-contamination of the bacteroid and plant fractions resulted in estimates that 36.2% of the total adenylate pool was in bacteroids, and 59.4% was in the plant fraction of the central zone. These are the first quantitative assessments of adenylate distribution in the plant and bacteroid fractions of legume nodules. These estimates were combined with theoretical calculations of rates of ATP consumption in the cortex (9.5 nmol g-1 fresh weight of nodule s-1), plant central zone (38 nmol g-1 fresh weight of nodule s-1), and bacteroids (62 nmol g-1 fresh weight of nodule s-1) of soybean nodules to estimate the time constants for turnover of the total adenylate pool and the ATP pool within each nodule fraction. The low values for time constant (1.6-5.8 s for total adenylate, 0.9-2.5 s for ATP only) in each fraction reflect the high metabolic activity of soybean nodules and provide a background for further studies of the role of adenylates in O2-limited nodule metabolism.
Full Text
The Full Text of this article is available as a PDF (923.1 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bergersen F. J., Turner G. L. Leghaemoglobin and the supply of O2 to nitrogen-fixing root nodule bacteroids: presence of two oxidase systems and ATP production at low free O2 concentration. J Gen Microbiol. 1975 Dec;91(2):345–354. doi: 10.1099/00221287-91-2-345. [DOI] [PubMed] [Google Scholar]
- Ching T. M., Bergersen F. J., Turner G. L. Energy status, growth and nitrogenase activity in continuous cultures of Rhizobium sp. strain CB756 supplied with NH+4 and various rates of aeration. Biochim Biophys Acta. 1981 Jun 12;636(1):82–90. doi: 10.1016/0005-2728(81)90078-5. [DOI] [PubMed] [Google Scholar]
- Ching T. M. Regulation of nitrogenase activity in soybean nodules by ATP and energy charge. Life Sci. 1976 May 15;18(10):1071–1076. doi: 10.1016/0024-3205(76)90140-5. [DOI] [PubMed] [Google Scholar]
- Gerhardt R., Heldt H. W. Measurement of subcellular metabolite levels in leaves by fractionation of freeze-stopped material in nonaqueous media. Plant Physiol. 1984 Jul;75(3):542–547. doi: 10.1104/pp.75.3.542. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heckmann M. O., Drevon J. J., Saglio P., Salsac L. Effect of Oxygen and Malate on NO(3) Inhibition of Nitrogenase in Soybean Nodules. Plant Physiol. 1989 May;90(1):224–229. doi: 10.1104/pp.90.1.224. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hunt S., King B. J., Canvin D. T., Layzell D. B. Steady and nonsteady state gas exchange characteristics of soybean nodules in relation to the oxygen diffusion barrier. Plant Physiol. 1987 May;84(1):164–172. doi: 10.1104/pp.84.1.164. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hunt S., King B. J., Layzell D. B. Effects of gradual increases in o(2) concentration on nodule activity in soybean. Plant Physiol. 1989 Sep;91(1):315–321. doi: 10.1104/pp.91.1.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuzma M. M., Hunt S., Layzell D. B. Role of Oxygen in the Limitation and Inhibition of Nitrogenase Activity and Respiration Rate in Individual Soybean Nodules. Plant Physiol. 1993 Jan;101(1):161–169. doi: 10.1104/pp.101.1.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Upchurch R. G., Mortenson L. E. In vivo energetics and control of nitrogen fixation: changes in the adenylate energy charge and adenosine 5'-diphosphate/adenosine 5'-triphosphate ratio of cells during growth on dinitrogen versus growth on ammonia. J Bacteriol. 1980 Jul;143(1):274–284. doi: 10.1128/jb.143.1.274-284.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Walsh K. B., Vessey J. K., Layzell D. B. Carbohydrate supply and n(2) fixation in soybean : the effect of varied daylength and stem girdling. Plant Physiol. 1987 Sep;85(1):137–144. doi: 10.1104/pp.85.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wong P. P., Evans H. J. Poly-beta-hydroxybutyrate Utilization by Soybean (Glycine max Merr.) Nodules and Assessment of Its Role in Maintenance of Nitrogenase Activity. Plant Physiol. 1971 Jun;47(6):750–755. doi: 10.1104/pp.47.6.750. [DOI] [PMC free article] [PubMed] [Google Scholar]