Quantitative NMR microscopy of osmotic stress responses in maize and pearl millet.

The effect of osmotic stress (-0.35 MPa) on the cell water balance and apical growth was studied non-invasively for maize (Zea mays L., cv. LG 11) and pearl millet (Pennisetum americanum L., cv. MH 179) by (1)H NMR microscopy in combination with water uptake measurements. Single parameter images of the water content and the transverse relaxation time (T(2)) were used to discriminate between the different tissues and to follow the water status of the apical region during osmotic stress. The T(2) values of non-stressed stem tissue turned out to be correlated to the cell dimensions as determined by optical microscopy. Growth was found to be strongly inhibited by mild stress in both species, whereas the water uptake was far less affected. During the experiment hardly any changes in water content or T(2) in the stem region of maize were observed. In contrast, the apical tissue of pearl millet showed a decrease in T(2) within 48 h of stress. This decrease in T(2) is interpreted as an increase in the membrane permeability for water.

Triplet state magnetic resonance and fluorescence spectroscopy of metal-substituted hemoglobins.

Fluorescence detected magnetic resonance (FDMR) spectra detected at 596 nm of zinc-substituted hemoglobins at 4.2 K show a split D-E transition, which is not observed for zinc protoporphyrins ligated by methylimidazole in glasses. Incorporation of the zinc heme into the globin pocket is also accompanied by a blue shift of the fluorescence of 20 nm at 4.2 K. FDMR spectra recorded at 576 nm do not show the D-E splitting. The D-E splitting and the huge blue shift are not observed for the magnesium-substituted hemoglobins. Fluorescence measurements at 4.2 K and 77 K, and EPR measurements at 110 K, were carried out to obtain information about the ligation states of the zinc and magnesium protoporphyrins in glasses and in hemoglobin. The results are explained by considering ligation effects and distortion of the porphyrin plane.

Fluorescence detected magnetic resonance (FDMR) of green sulfur photosynthetic bacteria Chlorobium sp.

Fluorescence Detected Magnetic Resonance (FDMR) spectra have been measured for whole cells and isolated chlorosomal fractions for the green photosyntheic bacteria Chlorobium phaeobacteroides (containing bacteriochlorophyll e, and isorenieratene as major carotenoid) and Chlorobium limicola (containing bacteriochlorophyll c, and chlorobactene as major carotenoid). The observed transition at 237 MHz (identical in both bacteria) and > 1100 MHz can be assigned, by analogy with published data on other carotenoids, to the 2E and D + E transitions, respectively, of Chlorobium carotenoids. Their zero field splitting (ZFS) parameters are estimated to be: |D|=0.0332 cm(-1) and |E|=0.0039 cm(-1) (chlorobactene), and |D|=0.0355 cm(-1) and |E|=0.0039 cm(-1) (isorenieratene). In the intermediate frequency range 300-1000 MHz the observed transitions can be assigned to chlorosomal bacteriochlorophylls c and e, and to bacteriochlorophyll a located in the chlorosome envelope and water-soluble protein. The bacteriochlorophyll e triplet state measured in 750 nm fluorescence (aggregated chlorosomal BChl e) is characterised by the ZFS parameters: |D|=0.0251 cm(-1) and |E|=0.0050 cm(-1).

Quantitative measurement and imaging of transport processes in plants and porous media by 1H NMR.

NMR and MRI have been applied to transport processes, that is, net flow and diffusion/perfusion, of water in whole plants, cells, and porous materials. By choosing proper time windows and pulse sequences, magnetic resonance imaging can be made selective for each of the two transport processes. For porous media and plant cells the evolution of the spatial distribution of excited spins has been determined by q-space imaging, using a 20 MHz pulsed 1H NMR imager. The results of these experiments are explained by including spin-relaxation and exchange at boundaries. A 10 MHz portable 1H NMR spectrometer is described, particularly suitable to study the response of net flow in plants and canopies to changing external conditions.

Fluorescence detected magnetic resonance of the primary donor and inner core antenna chlorophyll in Photosystem I reaction centre protein: Sign inversion and energy transfer.

The Photosystem I reaction centre protein CP1, isolated from barley using polyacrylamide gel electrophoresis showed an EPR (Electron Paramgnetic Resonance) spectrum with the polarisation pattern AEEAAE, typical of the primary donor triplet state (3)P700, created via radical pair formation and recombination. (3)P700 could also be detected by Fluorescence Detected Magnetic Resonance (FDMR) at λf > 700 nm even in the presence of a large number of chlorophyll antennae. Its zero field splitting parameters, D=282.5×10(-4) cm(-1) and E=38.5×10(-4) cm(-1), were independent of the detection wavelength, and agreed with ADMR (Absorption Detected Magnetic Resonance) and EPR values. The signs of the (3)P700 D+E and D-E transitions were positive (increase in fluorescence intensity on applying a resonance microwave field). In contrast, in the emission band 685 < λf < 700 nm FDMR spectra with negative D+E and D-E transitions were detected, and the D value was wavelength-dependent. These FDMR results support an excitation energy transfer model for CP1, derived from time-resolved fluorescence studies, in which two chlorophyll antenna forms are distinguished, with fluorescence at 685 < λf < 700 nm (inner core antennae, F690), and λf > 700 nm (low energy antenna sites, F720), in addition to the P700. The FDMR spectrum in F690 emission can be interpreted as that of (3)P700, observed via reverse singlet excitation energy transfer and added to the FDMR spectrum of the antenna triplet states generated via intramolecular intersystem crossing. This would indicate that reversible energy transfer between F690 and P700 occurs even at 4.2 K.

Description of enzyme kinetics in reversed micelles. 1. Theory.

In the literature measurements of kinetic data of enzymes in reversed micelles have been interpreted in two ways. In the first, all enzyme parameters are expressed with respect to the total volume of the reversed micellar solution. In the second, the enzymatic conversion is related only to the fraction of the volume consisting of aqueous solution (pseudophase model). In this paper equations are derived describing the rate of an enzymatic reaction for three different kinds of enzymes: enzymes obeying Michaelis-Menten kinetics, enzymes following a ping-pong bi-bi mechanism and enzymes which convert substrates according to an ordered mechanism. In deriving these equations, a distinction is made between intermicellar exchange reactions of substrate(s) and product(s) and the enzymatic reaction which takes place in the waterpool of a reversed micelle. In the description, all intrinsic rate constants of the enzyme are assumed to be independent of its environment. The rate equations show that the presence and efficiency of the intermicellar exchange reaction, which supplies the enzyme with substrate and removes product, can affect the rate of an enzymatic reaction under common experimental conditions. Whereas kinetic parameters derived from double-reciprocal plots often seem to be affected by enclosure in reversed micelles, these apparent deviations from kinetics in aqueous media can be explained by the model presented here as arising from exchange phenomena. Neither the experimentally determined maximum enzyme velocity, vmax, nor the Michaelis constants are affected by the incorporation of the enzyme in reversed micelles. The deviations of kinetic parameters from the aqueous values are shown to depend strongly on the concentration of reversed micelles, the intermicellar exchange rate and the volume fraction of water, a dependence in agreement with findings reported in the literature.

Enzyme kinetics in reversed micelles. 2. Behaviour of enoate reductase.

Enoate reductase (EC 1.3.1.31) can stereospecifically reduce a variety of alpha,beta-unsaturated carboxylates. Its use was extended to apolar media by incorporating the enzyme into a reversed micellar medium. The kinetics of the enzyme in such a medium have been investigated using 2-methylbutenoic acid as substrate and NADH as a cofactor and compared with the reaction rates in aqueous solution. In aqueous solution the enzyme obeys a ping pong mechanism [Bühler et al. (1982) Hoppe-Seyler's Z. Physiol. Chem 363, 609-625]. In 50 mM Hepes pH = 7.0 with ionic strength of 0.05 M the Michaelis constants for NADH and 2-methylbutenoic acid are 20 microM and 6.0 mM respectively. In reversed micelles the kinetics of the reaction (Michaelis constant, maximum velocity as well as inhibitory effects) were markedly different. The rate of the enzymatic reaction of enoate reductase was studied using various concentrations of 2-methylbutenoic acid and various NADH concentrations. In reversed micelles composed of the anionic detergent sodium di(ethylhexyl)sulphosuccinate, the enzymatic reaction deviates substantially from the values in aqueous solution. Using our model (see preceding paper in this issue of the journal), all kinetics could be explained as evolving from enclosure in reversed micelles without any change in the intrinsic rate parameters of the enzyme. So the enzyme itself is unaffected by incorporation in reversed micelles, but the rate of intermicellar exchange as well as the microheterogeneity of the medium, resulting in very high local concentrations of the substrate, are the most important factors altering the reaction pattern. The effect of the composition of the reversed micellar medium was also investigated using either a nonionic or a cationic surfactant. In these solutions too, exchange and microheterogeneity of the medium proved to be the most important parameters influencing the enzymatic reaction. In all reversed micellar solutions inhibition by the enoate was observed at an overall concentration of 0.5-5 mM, implying that a concentration of substrate equal to the Km value in aqueous solution may already cause inhibition in reversed micelles. At this level no inhibition by NADH was observed. The microheterogeneity of the medium also explains this inhibition of the enzyme at relatively low 2-methylbutenoic acid concentrations.

500 MHz 1H NMR of chlorophylls in the major light-harvesting chlorophyll-protein complex of photosystem II.

500 MHz 1H NMR spectra were obtained of solutions containing oligomeric and monomeric forms of Chl a/b-P2, the major light-harvesting chlorophyll a/b-protein complex of photosystem II, isolated from thylakoid membranes of barley (Hordeum vulgare). Oligomers showed only a broad unresolved spectrum, but for monomers several downfield-shifted chlorophyll proton resonances were observed, assigned to the alpha and beta methine protons and the formyl proton of Chl-b. Identifying the observed shifts as ring-current shifts, these NMR data can be matched with previously obtained optical data confirming the trimeric arrangement of Chl-b in Chl a/b-P2 protein, with a distance between the chromophore centers of approximately 12 A.

Substrate Utilization by Suspension Cultures and Somatic Embryos of Daucus carota L. Measured by C NMR.

The uptake and utilization of sucrose by embryogenic suspension cultures of carrot (Daucus carota L.) growing in the presence of 2,4-D and by somatic embryos derived from these cultures was monitored using (13)C nuclear magnetic resonance. The exogeneously supplied sucrose was completely hydrolyzed before cell entry; glucose was taken up preferentially when the cells were cultured in the presence of 2,4-D, while glucose and fructose were utilized at similar rates by somatic embryos in the absence of 2,4-D. Both suspension cells and somatic embryos accumulated high intracellular levels predominantly of glucose and sucrose, the latter being resynthesized intracellularly from the constitutive hexoses. Initially, fructose was converted mainly into glucose and sucrose rather than being catabolized directly through glycolysis or the pentose phosphate pathway. Carbohydrate supply that exceeded cellular demand resulted in intracellular accumulation of mono- or disaccharides. The capacity of cultured carrot cells to produce somatic embryos appeared to be positively correlated with high intracellular levels of glucose.

Noninvasive measurement of plant water flow by nuclear magnetic resonance.

Water flow through the stem of an intact cucumber plant has been measured by using pulsed NMR. This method yields the linear flow velocity of the sapstream, found to be proportional to the loss of weight due to evaporation. The presence of a large excess of stationary water (for cucumber 95% of the total water content) does not interfere with the detection of a small amount of flowing water, due to cancellation of the NMR signal of stationary water. This makes the method particulary suitable for application to biological systems with a high stationary water content.

Magic angle spinning carbon-13 NMR of tobacco mosaic virus. An application of the high-resolution solid-state NMR spectroscopy to very large biological systems.

Magic angle spinning 13C NMR was used to study tobacco mosaic virus (TMV) in solution. Well-resolved 13C NMR spectra were obtained, in which several carbon resonances of amino acids of the TMV coat protein subunits that are not observable by conventional high-resolution NMR spectroscopy can be designed. RNA resonance were absent, however, in the magic angle spinning 13C NMR spectra. Since three different binding sites are available for each nucleotide of the RNA, this is probably due to a line broadening caused by distributions of isotropic chemical shift values. In 13C-enriched TM 13C-13C dipolar interactions also gave rise to line broadening. By suitable pulse techniques that discriminate carbon resonances on the basis of their T1 and T1 rho values, it was possible to select particular groups of carbon nuclei with characteristic motional properties. Magic angle spinning 13C NMR spectra obtained with these pulse techniques are extremely well resolved.

[1H]Spin-echo nuclear magnetic resonance in plant tissue. I. The effect of Mn(II) and water content in wheat leaves.

The effect of age-dependent Mn(II)-gradients, as observed by electron paramagnetic resonance (EPR), on the [1H]NMR spin-spin relaxation time (T2) was studied in wheat leaves. A non-exponential T2 spin-echo decay was always observed, revealing the presence of at least two different fractions of non- (or slowly) exchanging water in the leaves. No effect of the Mn(II)-concentration on T2 of the separate water fractions (covering approximately 90% of the total water content) has been found. From these observations we conclude that Mn(II) is present in bound form. The dependence of T2 on water content can be explained with a two-state model, demonstrating the occurrence of fast exchange within each of the two slowly exchanging water fractions.

Magnetic field effects on radical pair intermediates in bacterial photosynthesis.

We have investigated the effects of magnetic fields on the formation and decay of excited states in the photochemical reaction centers of Rhodopseudomonae sphaeroides. In chemically reduced reaction centers, a magnetic field decreases the fraction of the transient state PF that decays by way of the bacteriochlorophyll triplet state PR. At room temperature, a 2-kG field decreases the quantum yield of Pr by about 40%. In carotenoid-containing reaction centers, the yield of the carotenoid triplet state which forms via PR is reduced similarly. The effect of the field depends monotonically on field-strength, saturating at about 1 kG. The effect decreases at lower temperatures, when the yield of PR is higher. Magnetic fields do not significantly affect the formation of the triplet state of bacteriochlorophyll in vitro, the photooxidation of P870 in reaction centers at moderate redox potential, or the decay kinetics of states PF and PR. The effect of magnetic fields support in view that state PF is a radical pair which is born in a singlet state but undergoes a rapid transformation into a mixture of singlet and triplet states. A simple kinetic model can account for the effects of the field and relate them to the temperature dependence of the yield of PR.