In vivo 133Cs-NMR a probe for studying subcellular compartmentation and ion uptake in maize root tissue.

Three 133Cs-NMR signals were observed in the spectra of CsCl-perfused and CsCl-grown maize seedling root tips. Two relatively broad lower field resonances were assigned to the subcellular, compartmented Cs+ in the cytoplasm and vacuole, respectively. The rate of area increase of the broader cytoplasmic Cs resonance was about 9-times faster than that of the vacuolar signal during the first 300 min of tissue perfusion with CsCl. In addition, the spin lattice relaxation time of the cytoplasmic Cs resonance was approx. 3-times shorter than that of the extracellular resonance, while the Cs+ signal associated with the metabolically less active vacuolar compartment exhibited a relaxation time comparable to that of the extracellular signal. 133Cs spectra of excised, maize root tips and excised top sections of the root adjacent to the kernel, each grown in 10 mM CsCl showed a difference in the relative areas of the Cs resonance corresponding to the distinct cytoplasm/vacuole volume ratio of these well differentiated sections of the root. The high correlation of counterion concentration with 133Cs chemical shifts suggested that the larger downfield shift exhibited by the cytoplasmic confined Cs+ was due principally to the higher ionic strength and protein content in this compartment. Such observations indicate that 133Cs-NMR might be employed for studying ionic strength, and osmotic pressure associated chemical shifts and the transport properties of Cs+ (perhaps as an analogue for K+) in subcellular compartments of plant tissues.

Structural studies of a phosphocholine substituted beta-(1,3);(1,6) macrocyclic glucan from Bradyrhizobium japonicum USDA 110.

In our previous in vivo 31P study of intact nitrogen-fixing nodules (Rolin, D.B., Boswell, R.T., Sloger, C., Tu, S.I. and Pfeffer, P.E., 1989 Plant Physiol. 89, 1238-1246), we observed an unknown phosphodiester. The compound was also observed in the spectra of isolated bacteroids as well as extracts of the colonizing Bradyrhizobium japonicum USDA 110. In order to characterize the phosphodiester in the present study, we took advantage of the relatively hydrophobic nature of the material and purified it by elution from a C-18 silica reverse-phase chromatography column followed by final separation on an aminopropyl silica HPLC column. Structural characterization of this compound with a molecular weight of 2271 (FAB mass spectrometry), using 13C-1H and 31P-1H heteronuclear 2D COSY and double quantum 2D phase sensitive homonuclear 1H COSY NMR spectra, demonstrated that the molecule contained beta-(1,3); beta-(1,6); beta-(1,3,6) and beta-linked non-reducing terminal glucose units in the ratio of 5:6:1:1, respectively, as well as one C-6 substituted phosphocholine (PC) moiety associated with one group of (1,3) beta-glucose residues. Carbohydrate degradation analysis indicated that this material was a macrocyclic glucan, (absence of a reducing end group) with two separated units containing three consecutively linked beta-(1,3) glucose residues and 6 beta-(1,6) glucose residues. The sequences of beta-(1,3)-linked glucose units contained a single non-reducing, terminal, unsubstituted glucose linked at the C-6 position and a PC group attached primarily to an unsubstituted C-6 position of a beta-(1,3)-linked glucose.

In vivo 13C NMR study of the bidirectional reactions of the Wood-Werkman cycle and around the pyruvate node in Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici.

This study used in vitro 13C NMR spectroscopy to directly examine bidirectional reactions of the Wood-Werkman cycle involved in central carbon metabolic pathways of dairy propionibacteria during pyruvate catabolism. The flow of [2-13C]pyruvate label was monitored on living cell suspensions of Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici under acidic conditions. P. shermanii and P. acidipropionici cells consumed pyruvate at apparent initial rates of 161 and 39 micromol min(-1) g(-1) (cell dry weight), respectively. The bidirectionality of reactions in the first part of the Wood-Werkman cycle was evident from the formation of intermediates such as [3-13C]pyruvate and [3-13C]malate and of products like [2-13C]acetate from [2-13C]pyruvate. For the first time alanine labeled on C2 and C3 and aspartate labeled on C2 and C3 were observed during [2-13C]pyruvate metabolism by propionibacteria. The kinetics of aspartate isotopic enrichment was evidence for its production from oxaloacetate via aspartate aminotransferase. Activities of a partial tricarboxylic acid pathway, acetate synthesis, succinate synthesis, gluconeogenesis, aspartate synthesis, and alanine synthesis pathways were evident from the experimental results.

In vivo nuclear magnetic resonance study of the osmoregulation of phosphocholine-substituted beta-1,3;1,6 cyclic glucan and its associated carbon metabolism in Bradyrhizobium japonicum USDA 110.

A phosphocholine-substituted beta-1,3;1,6 cyclic glucan (PCCG), an unusual cyclic oligosaccharide, has been isolated from Bradyrhizobium japonicum USDA 110 (D. B. Rolin, P. E. Pfeffer, S. F. Osman, B. S. Swergold, F. Kappler, and A. J. Benesi, Biochim. Biophys. Acta 1116:215-225, 1992). Data presented here suggest that PCCG synthesis is dependent on the carbon metabolism and that osmotic regulation of its biosynthesis parallels regulation of membrane-derived oligosaccharide biosynthesis observed in Escherichia coli (E. P. Kennedy, M. K. Rumley, H. Schulman, and L. M. G. van Golde, J. Biol. Chem. 251:4208-4213, 1976) and Agrobacterium tumefaciens (G. A. Cangelosi, G. Martinetti, and E. W. Nester, J. Bacteriol. 172:2172-2174, 1990). Growth of B. japonicum USDA 110 cells in the reference medium at relatively low osmotic pressures (LO) (65 mosmol/kg of H2O) caused a large accumulation of PCCG and unsubstituted beta-1,3;1,6 cyclic glucans (CG). Sucrose and polyethylene glycol, nonionic osmotica, reduce all growth rates and inhibit almost completely the production of PCCG at high osmotic pressures (HO) above 650 and 400 mosmol/kg of H2O), respectively. We used in vivo 13C nuclear magnetic resonance spectroscopy to identify the active osmolytes implicated in the osmoregulation process. The level of alpha,alpha-trehalose in B. japonicum cells grown in autoclaved or filter-sterilized solutions remained constant in HO (0.3 M sucrose or 250 g of polyethylene glycol 6000 per liter) medium. Significant amounts of glycogen and extracellular polysaccharides were produced only when glucose was present in the autoclaved HO 0.3 M sucrose media. The results of hypo- and hyperosmotic shocking of B. japonicum USDA 110 cells were monitored by using in vivo 31P and 13C nuclear magnetic resonance spectroscopy. The first observed osmoregulatory response of glycogen-containing cells undergoing hypoosmotic shock was release of P(i) into the medium. Within 7 h, reabsorption of P(i) was complete and production of PCCG was initiated. After 12 h, the PCCG content had increased by a factor of 7. Following the same treatment, cells containing little or no glycogen released trehalose and failed to produce PCCG. Thus the production of PCCG/CG in response to hypoosmotic shocking of stationary-phase cells was found to be directly linked to the interconversion of stored glycogen. Hyperosmotic shocking of LO-grown stationary-phase cells with sucrose had no effect on the content of previously synthesized CG/PCCG. The PCCG/CG content and its osmotically induced biosynthesis are discussed in terms of carbon metabolism and a possible role in hypoosmotic adaptation in B. japonicum USDA 110.

In VivoP NMR Spectroscopic Studies of Soybean Bradyrhizobium Symbiosis: I. Optimization of Parameters.

(31)P NMR spectroscopy was used to study in vivo the symbiotic state established between soybean (Glycine max [L.] Merr. cv Williams) and Bradyrhizobium japonicum (USDA 110 and 138). Different experimental conditions were used to maintain perfused, respiring detached or attached nodules in an NMR magnet. The pH of the perfusion medium affected the cytoplasmic pH and the resolution of the spectra. The internal Pi content and distribution were assessed as a function of nodule age and green-house growth conditions and the rate of glucose and 2-deoxyglucose uptake into nodules in split and intact states. The major metabolites (glucose-6-P, fructose-1,6-diP, P-choline, Pi, NTP, UDP-glc, and NAD) were readily identified from (31)P NMR spectra of perchloric acid extracts of nodules with the exception of one unknown phosphorus metabolite. Nodules stressed by glucose deprivation demonstrated movement of Pi between the vacuole and cytoplasmic compartments not previously observed in (31)P NMR studies.

P relaxation responses associated with n(2)/o(2) diffusion in soybean nodule cortical cells and excised cortical tissue.

N(2)-fixing Bradyrhizobium japonicum nodules and cortical tissue derived from these nodules were examined in vivo by (31)P nuclear magnetic resonance (NMR) spectroscopy. Perfusion of the viable nodules and excised cortical tissue with O(2) followed by N(2) or Ar caused a loss of orthophosphate (Pi) resonance magnetization associated with the major portion of acidic Pi (delta 0.9 ppm, pH 5.5) residing in the cortical cells. Resumption of O(2) perfusion restored approximately 80% of the intensity of this peak. Detailed examination of the nuclear relaxation processes, spin-lattice relaxation time (T(1)), and spin-spin relaxation time (T(2)), under perfusion with N(2) or Ar as opposed to O(2), indicated that loss of signal was due to T(1) saturation of the acidic Pi signal under the rapid-pulsed NMR recycling conditions. In excised cortical tissue, Pi T(1), values derived from biexponential relaxation processes under perfusing O(2) were 59% 3.72 +/- 0.93 s and 41% 0.2 +/- 0.08 s, whereas under N(2) these values were 85% 7.07 +/- 1.36 s and 15% 0.39 +/- 0.07 s. The T(1) relaxation behavior of whole nodule vacuolar Pi showed the same trend, but the overall values were somewhat shorter. T(2) values for cortical tissue were also biexponential but were essentially the same under O(2) (38% 0.066 +/- 0.01 s and 63% 0.41 +/- 0.08 s) and N(2) (39% 0.07 +/- 0.01 s and 61% 0.37 +/- 0.01 s) perfusion. Soybean (Glycine max) root tissue as well as Pi solutions exhibited single exponential T(1) decay values that were not altered by changes in the perfusing gas. These data indicate that oxygen induces a change in the physical environment of phosphate in the cortical cell tissue. Although under certain conditions oxygen has been observed to act as a paramagnetic relaxation agent, model T(1) experiments demonstrate that O(2) does not significantly influence Pi relaxation in this manner. Alternatively, we suggest that an increase in solution viscosity brought on by the production of an occlusion glycoprotein (under O(2) perfusion) is responsible for the observed relaxation changes.

Observation of the Oxygen Diffusion Barrier in Soybean (Glycine max) Nodules with Magnetic Resonance Microscopy.

The effects of selected gas perfusion treatments on the spinlattice relaxation times (T(1)) of the soybean (Glycine max) nodule cortex and inner nodule tissue were studied with (1)H high resolution magnetic resonance microscopy. Three gas treatments were used: (a) perfusion with O(2) followed by N(2); (b) O(2) followed by O(2); and (c) air followed by N(2). Soybean plants with intact attached nodules were placed into the bore of a superconducting magnet and a selected root with nodules was perfused with the gas of interest. Magnetic resonance images were acquired with repetition times from 50 to 3200 ms. The method of partial saturation was used to calculate T(1) times on selected regions of the image. Calculated images based on T(1) showed longer T(1) values in the cortex than in the inner nodule during all of the gas perfusions. When nodules were perfused with O(2)-O(2), there was no significant change in the T(1) of the nodule between the two gas treatments. When the nodule was perfused with O(2)-N(2) or air-N(2), however, the T(1) of both the cortex and inner nodule increased. In these experiments, the increase in T(1) of the cortex was 2- to 3-fold greater than the increase observed in the inner nodule. A similar change in T(1) was found in detached live nodules, but there was no change in T(1) with selective gas perfusion of detached dead nodules. These observations suggest that cortical cells respond differently to selected gas perfusion than the inner nodule, with the boundary of T(1) change sharply delineated at the interface of the inner nodule and the inner cortex.