N2-fixing Bradyrhizobium japonicum nodules and cortical tissue derived from these nodules were examined in vivo by 31P nuclear magnetic resonance (NMR) spectroscopy. Perfusion of the viable nodules and excised cortical tissue with 02 followed by N2 or Ar caused a loss of orthophosphate (Pi) resonance magnetization associated with the major portion of acidic Pi (5 0.9 ppm, pH 5.5) residing in the cortical cells. Soybean nodules contain three cortical cell layers (44% of the nodule volume) (28), an outer cortex, an endodermis, and an inner cortex. The inner cortex functions as a diffusion barrier to balance inward oxygen flux with bacteroid respiratory oxygen demand (15,20,31,32). Tjepkema and Yocum (29) and later Witty et al. (34) verified that the oxygen concentration decreased across the cortical layer until it reached a minimum at the inner cortex, the presumed major barrier to gas diffusion. Numerous investigators (2,7,8,11,27,28) have examined the diffusion of 02 through this barrier and have proposed that the sites of entry are air spaces filled with water the depth of which can be varied through expansion or contraction of the adjacent inner cortex cells. On the other hand, Witty and coworkers (26, 34) have speculated that cell expansion into the intercellular space is responsible for restricting the passage of 02 into the inner nodule in response to diminished respiration. VandenBosch et al. (30) and Bradley et al. (3) observed a unique glycoprotein in the infection thread matrix and intercellular space of pea nodules. Further investigation of this material in soybean nodules by James and coworkers (9) demonstrated that its elaboration was greatly enhanced in the presence of oxygen. These authors (9) hypothesized that this 'occlusion protein' is responsible for increasing 02 diffusion resistance in the cortical cell layer by blocking the intercellular air spaces.At present the nature of the oxygen-regulating miechanism(s) associated with the cortical layer is controversial. In this and the accompanying paper (12), we describe relaxation data obtained from 31P NMR spectroscopy and 'H magnetic resonance imaging studies that suggest that increasing viscosity resulting from the production of cortical cell glycoprotein may be associated with enhanced 02 diffusion resistance in soybean (Glycine max) nodules.
MATERIALS AND METHODS