Abstract
A method is presented for the rapid measurement of the spectral properties of detached nodules of pea (Pisum sativum L. cv “Sparkle”) by diffuse reflectance spectroscopy. After correcting the spectra for surface light scattering, the spectrum of leghemoglobin is obtained. From this, the fractional oxygenation of leghemoglobin and the internal O2 concentration can be calculated. With this method, we determined internal O2 while measuring nitrogenase activity (C2H2) in detached pea nodules over a range of external O2 concentrations. Nitrogenase activity was maximum when leghemoglobin was 25% oxygenated, corresponding to a calculated free O2 concentration of 45 nanomolar in infected cells. Advantages of this method over previous methods which employed transmitted light are: (a) many nodules can be assayed simultaneously, (b) nitrogenase activity (C2H2) can be determined at the same time as spectra are recorded, and (c) spectra can be obtained from nodules submerged in buffer containing metabolic effectors.
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- APPLEBY C. A. The oxygen equilibrium of leghemoglobin. Biochim Biophys Acta. 1962 Jul 2;60:226–235. doi: 10.1016/0006-3002(62)90398-0. [DOI] [PubMed] [Google Scholar]
- Appleby C. A. Properties of leghaemoglobin in vivo, and its isolation as ferrous oxyleghaemoglobin. Biochim Biophys Acta. 1969;188(2):222–229. doi: 10.1016/0005-2795(69)90069-5. [DOI] [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]
- King B. J., Hunt S., Weagle G. E., Walsh K. B., Pottier R. H., Canvin D. T., Layzell D. B. Regulation of o(2) concentration in soybean nodules observed by in situ spectroscopic measurement of leghemoglobin oxygenation. Plant Physiol. 1988 Jun;87(2):296–299. doi: 10.1104/pp.87.2.296. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kobrick J. L., Witt C., Miletti J. An automated portable night vision testing system. Aviat Space Environ Med. 1987 Nov;58(11):1127–1129. [PubMed] [Google Scholar]
- Monroe J. D., Larue T. A. Effects of carbohydrate on the internal oxygen concentration, oxygen uptake, and nitrogenase activity in detached pea nodules. Plant Physiol. 1989 Oct;91(2):603–609. doi: 10.1104/pp.91.2.603. [DOI] [PMC free article] [PubMed] [Google Scholar]