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.     

Diffusion kurtosis imaging in head and neck cancer: A correlation study with dynamic contrast enhanced MRI

PurposeTo investigate the biophysical meaning of Diffusion Kurtosis Imaging (DKI) parameters via correlations with the perfusion parameters obtained from a long Dynamic Contrast Enhanced MRI scan, in head and neck (HN) cancer.MethodsTwenty two patients with newly diagnosed HN tumor were included in the present retrospective study. Some patients had multiple lesions, therefore a total of 26 lesions were analyzed. DKI was acquired using 5b values at 0, 500, 1000,1500 and 2000 s/mm². DCE-MRI was obtained with 130 dynamic volumes, with a temporal resolution of 5 s, to achieve a long scan time (>10 min). The apparent diffusion coefficient D_app and apparent diffusional kurtosis K_app were calculated voxel-by-voxel, removing the point at b value = 0 to eliminate possible perfusion effects on the parameter estimations. The transfer constants K^trans and K_ep, v_e, and the histogram-based entropy (En) and interquartile range (IQR) of each DCE-MRI parameter were quantified. Correlations between all variables were investigated by the Spearman’s Rho correlation test.ResultsModerate relationships emerged between D_app and K_ep (Rho =  − 0.510, p = 0.009), and between D_app and v_e (Rho = 0.418, p = 0.038). En(K_ep) was significantly related to K_app (Rho = 0.407, p = 0.043), while IQR(K_ep) showed an inverse association with D_app (Rho = -0.422, p = 0.035).ConclusionsA weak to intermediate correlation was found between DKI parameters and both K_ep and v_e. The kurtosis was associated to the intratumoral heterogeneity and complexity of the capillary permeability, expressed by En(K_ep).     

A proton nuclear magnetic resonance study of the physical changes in growing plant cell walls

Changes in broadline proton nuclear magnetic resonance parameters of cell walls during growth of etiolated hypocotyls of bean (Phaseolus vulgaris L.) indicate that cell wall structure becomes more rigid during development. Most of the changes are completed in the first 6 cm below cotyledon insertion and are correlated with increased restriction of proton movements in regions of dense polymer packing.Abbreviations FID free induction decay - M₂ second moment - M_2interpair interpair second moment - NMR nuclear magnetic resonance - T_1D dipolar relaxation time - T₂ spin-spin relaxation time This work was supported by grants from Natural Sciences and Engineering Research Council of Canada to A.L.M., I.E.P.T. and M. Bloom.     

Nuclear magnetic resonance relaxation times and plasmalemma water exchange in ivy bark

Measurement of nuclear magnetic resonance (NMR) relaxation times (transverse [T₂] and longitudinal [T₁]) for Hedera helix L. cv. Thorndale (ivy) bark water indicates the presence of at least two populations of water with different relaxation characteristics. One population of water with short T₂ and T₁ was found to be composed of both hydration water and extracellular free water. The second population of water with long T₂ and T₁ was identified as intracellular bulk water.     

Diffusion properties of bacteriophages through agarose gel membrane

A simple two‐chamber diffusion method was developed to study the diffusion properties of bacteriophages (phages). The apparent diffusion coefficients (D_app) of Myoviridae phage T4 and filamentous phage fNEL were investigated, and the diffusion of the phages was found to be much slower than the diffusion of three antibiotics, ciprofloxacin, penicillin G, and tetracycline. D_app of T4 and fNEL in water through filter paper were calculated to be 2.8 × 10^?11 m²/s and 6.8 × 10^?12 m²/s, respectively, and D_app of fNEL through agarose gel membrane, an artificial biofilm, was also calculated to be smaller than that of T4. In addition, D_app of phages through agarose gel was dependent on agarose concentration due to the similar size of phage and agarose gel mesh. We concluded that D_app of phages through an artificial biofilm is dependent on both phage morphology and biofilm density, and suggest the use of this method to study diffusion properties through real biofilms. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Nuclear Magnetic Resonance Evidence using D2O for Structured Water in Muscle and Brain

The electric quadrupole moment of the deuterium nucleus provides a nuclear magnetic resonance (NMR) probe of electric field gradients, and thereby of organization of tissue water. 8-17% of H₂O in rat muscle and brain was replaced by D₂O from 50% deuterated drinking water. The peak height of the steady-state NMR spectrum of D in muscle water was 74% lower than that of an equal concentration of D₂O in liquid water. Longitudinal NMR relaxation times (T₁) of D in water of muscle and brain averaged 0.092 and 0.131 sec, respectively, compared with 0.47 sec in D₂O in liquid water. Transverse NMR relaxation times (T₂) averaged 0.009 and 0.022 sec in D₂O of muscle and brain, respectively, compared with 0.45 sec in D₂O in liquid water. These differences cannot be explained by paramagnetic ions or by magnetic inhomogeneities, which leaves increased organization of tissue water as the only tenable hypothesis. Evidence was also obtained that 27% of muscle water and 13% of brain water exist as a separate fraction with T₂ of D₂O less than 2 × 10^-3 sec, which implies an even higher degree of structure. Each of the two fractions may consist of multiple subfractions of differing structure.     

Direct assessment of water exchange on a Gd(III) chelate bound to a protein

 The Gd(III) complex of 4-pentylbicyclo[2.2.2]octane-1-carboxyl-di-l-aspartyl-lysine-derived DTPA, [GdL(H₂O)]^2–, binds to serum albumin in vivo, through hydrophobic interaction. A variable temperature ¹⁷O NMR, EPR, and Nuclear Magnetic Relaxation Dispersion (NMRD) study resulted in a water exchange rate of k ²⁹⁸ _ex=4.2×10⁶ s^–1, and let us conclude that the GdL complex is identical to [Gd(DTPA)(H₂O)]^2– in respect to water exchange and electronic relaxation. The effect of albumin binding on the water exchange rate has been directly evaluated by ¹⁷O NMR. Contrary to expectations, the water exchange rate on GdL does not decrease considerably when bound to bovine serum albumin (BSA); the lowest limit can be given as k _{ex, GdL-BSA}=k _ex, GdL / 2. In the knowledge of the water exchange rate for the BSA-bound GdL complex, the analysis of its NMRD profile at 35  °C yielded a rotational correlation time of 1.0 ns, one order of magnitude shorter than that of the whole protein. This value is supported by the longitudinal ¹⁷O relaxation rates. This indicates a remarkable internal flexibility, probably due to the relatively large distance between the protein- and metal-binding moieties of the ligand. Received: 25 June 1998 / Accepted: 11 August 1998     

Magnetic resonance imaging (MRI) of water during cold acclimation and freezing in winter wheat

Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) were used to analyse changes in the physical state of water in wheat crowns during cold acclimation and during the freezing/thawing cycle. Spectroscopically measured average spin-spin relaxation times (T₂) decreased during cold acclimation and increased when plants were grown at normal temperature. Spin-spin relaxation images whose contrast is proportional to T₂, times were calculated allowing association of water relaxation with regions of tissue in spin-echo images during acclimation and freezing. Images taken during freezing revealed nonuniform freezing of tissue in crowns and roots. Acclimated and non-acclimated wheat crowns were imaged during freezing and after thawing. Spin-echo image signal intensity and T₂ times decreased dramatically between -4°C and -8°C as a result of a decrease in water mobility during freezing. Images collected during thawing were diffuse with less structure and relaxation times were longer, consistent with water redistribution in tissue after membrane damage.     

Water transport in maize roots under the influence of mercuric chloride and water stress: a role of water channels

The influence of inhibitor of water channels, HgCl₂, on water diffusion in maize (Zea mays L.) seedling roots was investigated with the pulsed nuclear magnetic resonance (NMR) method. Blocking of water channels decreased the water permeability of cell membranes by 1.5 – 2 times. This effect of HgCl₂ was exhibited only in the roots of seedlings grown in a nutrient solution containing Ca²⁺ and was reversed with Hg-scavenging agent β-mercaptoethanol. Subsequent incubation of Ca²⁺-deficient roots in the nutrient solution with Ca²⁺ recovered the sensitivity to HgCl₂. The water stress decreased water diffusion rates similarly to HgCl₂ and the effects of water stress and HgCl₂ were not additive. The obtained data demonstrate the possibilities of the pulsed NMR method for study of the transmembrane water exchange in vivo in connection with water channel functioning.     

QELS-SEF study on poly(lysine) in the extraordinary phase: Effect of electric field strength on the apparent diffusion coefficient

The technique of quasielastic light scattering (QELS) in the presence of a sinusoidal electric field (QELS-SEF) was used to examine the diffusion of poly(lysine) in the extraordinary phase, which is defined by the anomalously small value in the apparent diffusion coefficient (D_app, obtained by QELS methods) as the added salt concentration is lowered below 10 mM. Spectra were obtained for high-molecular-weight poly(lysine) (800,000) in 0.5 mM KCl, using a driving frequency of 90 Hz. It was observed that the linewidth, hence apparent diffusion coefficient (D_SF), increased with electric field strength (E) over the range 6 < E < 30 V/cm. As might be anticipated, the asymptotic limit, D_SF(E = 0), was almost numerically equivalent to D_app at this KCl concentration. As E approached the upper limit value of 31 V/cm, the value of D_SF approached the high salt (500 mM KCl) value of D_app. These observations are discussed in terms of small-ion dispersion effects.     

An NMR microscopic study of grape (Vitis vinifera L.)

Summary Mature healthy grape berries and berries wound-inoculated with the fungusBotrytis cinerea were examined by¹H NMR microimaging using 2D and 3D spin echo and gradient echo procedures. These NMR images were compared with representations obtained by conventional histology, where possible using the same specimens. 3D imaging datasets from excised seeds were reconstructed by surface rendering and maximum intensity projection to allow interpretation of their internal structure. T₂-weighted spin echo images revealed the major features of the pericarp, septum and loculi of whole berries. T₁-weighted images were less discriminatory of parenchyma tissues in the fruit but revealed the endosperm in seeds as a chemically shifted feature. A non-invasive study by T₁-weighted spin echo NMR imaging of infection byB. cinerea over a 6-day period showed that the disease spread throughout the exocarp but failed to spread in the mesocarp, a result confirmed by histological examination of the same specimen. Surface rendering of 3D datasets of excised seeds revealed the two ruminations of the endosperm and the distal location of the chalaza. The position of the embryonic axis was revealed in T₂-weighted maximum intensity projections. This noninvasive study revealed the need to apply a range of imaging techniques and parameters to visualise the structural features of the different parts of the grape berry.Abbrevations BF bright field - FDA fluorescein diacetate - FI field inhomogeneity - FOV field of view - NMR nuclear magnetic resonance - RF radiofrequency - T₁ spin-lattice relaxation time - T₂ spin-spin relaxation time - TE echo time - TMS tetramethylsilane - TR repeat time     

Thermodynamic analysis of the physical state of water during freezing in plant tissue, based on the temperature dependence of proton spin-spin relaxation

Multi-proton spin-echo images were collected from cold-acclimated winter wheat crowns (Triticum aestivum L.) cv. Cappelle Desprez at 400 MHz between 4 and ?4 °C. Water proton relaxation by the spin-spin (T2) mechanism from individual voxels in image slices was found to be mono-exponential. The temperature dependence of these relaxation rates was found to obey Arrhenius or absolute rate theory expressions relating temperature, activation energies and relaxation rates, Images whose contrast is proportional to the Arrhenius activation energy (E_a), Gibb's free energy of activation (ΔG^?), and the entropy of activation (ΔS^?) for water relaxation on a voxel basis were constructed by post-image processing. These new images exhibit contrast based on activation energies rather than rules of proton relaxation. The temperature dependence of water proton T₂ relaxation rates permits prediction of changes in the physical state of water in this tissue over modest temperature ranges. A simple model is proposed to predict the freezing temperature kof various tissue in wheat crowns. The average E_a and ΔH^? for water proton T₂ relaxation over the above temperature range in winter wheat tissue were ?6.4 ± 14.8 and ?8.6 ± 14.8kj mol^?1, respectively. This barrier is considerably lower than the E_a for proton translation in ice at 0°C, which is reported to be between 46.0 and 56.5 kj mol^?1     

Pearl millet transformation system using the positive selectable marker gene phosphomannose isomerase

Fertile transgenic pearl millet plants expressing a phosphomannose isomerase (PMI) transgene under control of the maize ubiquitin constitutive promoter were obtained using the transformation system described here. Proliferating immature zygotic embryos were used as target tissue for bombardment using a particle inflow gun. Different culture and selection strategies were assessed in order to obtain an optimised mannose selection protocol. Stable integration of the manA gene into the genome of pearl millet was confirmed by PCR and Southern blot analysis. Stable integration of the manA transgene into the genome of pearl millet was demonstrated in T₁ and T₂ progeny of two independent transformation events with no more than four to ten copies of the transgene. Similar to results obtained from previous studies with maize and wheat, the manA gene was shown to be a superior selectable marker gene for improving transformation efficiencies when compared to antibiotic or herbicide selectable marker genes.Abbreviations 2,4-D: 2,4-Diclorophenoxyacetic acid - IAA: Indole acetic acid - ICRISAT: International Crops Research Institute for the Semi-Arid Tropics - IZEs Immature zygotic embryos Communicated by H. Lörz     

Viability of Cururbita pepo pollen: biophysical and structural data

During ageing of the short-lived pollen grains of Cucurbita pepo L., water loss was examined in relation to viability using biophysical (¹H-nuclear magnetic resonance, NMR) and cytological methods (fluorochromatic reaction test, freezefracture and scanning electron microscopy). A semi-logarithmic representation of the pollen weight loss demonstrated the complexity of the dehydration process. A the study of proton loss using ¹H-NMR indicated that two major releases water of had taken place, each with different flux rates. Pulse ¹H-NMR experiments showed the occurrene of non-exponential signal decay as a function of time, indicating the existence of different fractions of water in a pollen grain sample. These fractions leave the pollen grain at different times during pollen dehydration, and one of them (that of the so-called vital water) can be related to pollen viability. The quantity of protons giving a signal during pulse ¹H-NMR experiments was very low when the pollen grains were judged to be dead according to the fluorochromatic test. Freeze-fracture replicas of these dead pollen grains (less than 25% water content) showed that the plasma membrane had become detached from the intine surface; this ultrastructural feature might therefore be involved in the loss of pollen viability.Abbreviations A initial amplitude of the NMR signal - A₂ quantity of water charcterized by T_2-2 - A₅ quantity of water characterized by T_2–5 - FCR fluorochromatic reaction - NMR nuclear magnetic resonance - T₂ transverse relaxation time - T_2-2 T₂ measured with 2 ms between each pulse of radiofrequency - T_2–5 T₂ measured with 5 ms between each pulse of radiofrequency     

Measurement of vulnerability to water stress‐induced cavitation in grapevine: a comparison of four techniques applied to a long‐vesseled species

Among woody plants, grapevines are often described as highly vulnerable to water‐stress induced cavitation with emboli forming at slight tensions. However, we found native embolism never exceeded 30% despite low xylem water potentials (Ψ_x) for stems of field grown vines. The discrepancy between native embolism measurements and those of previous reports led us to assess vulnerability curve generation using four separate methods and alterations (i.e. segment length and with/without flushing to remove embolism prior to measurement) of each. Centrifuge, dehydration and air‐injection methods, which rely on measurement of percentage loss of hydraulic conductivity (PLC) in detached stems, were compared against non‐invasive monitoring of xylem cavitation with nuclear magnetic resonance (NMR) imaging. Short segment air‐injection and flushed centrifuge stems reached >90 PLC at Ψ_x of‐0.5 and ?1.5 MPa, respectively, whereas dehydration and long‐segment air‐injection measurements indicated no significant embolism at Ψ_x > ?2.0 MPa. Observations from NMR agreed with the dehydration and long segment air‐injection methods, showing the majority of vessels were still water‐filled at Ψ_x > ?1.5 MPa. Our findings show V. vinifera stems are far less vulnerable to water stress‐induced cavitation than previously reported, and dehydration and long segment air‐injection techniques are more appropriate for long‐vesseled species and organs.     

Boundaries of the universal K3 region and plateau region of the dynamic structure factor for DNA

A sufficiently long semiflexible filamentous macromolecule is theroretically expected to exhibit three different domains of behavior of its apparent diffusion coefficient D_app(K) as a function of scattering vector K: (1) the small wave vector limit, where D_app(K) = D₀ is the translational diffusion coefficient of the center-of-mass; (2) the universal K³ region, where D_app(K) = (k_BT/6πη)K is a universal function of K independent of any property of the molecule itself; (c) the plateau region at large K², where D_app(K) approaches either a plateau, or gradually sloping quasiplateau, characteristic of local (elastic) rigid-body motions of the filament. The existence of each of these different domains has now been established experimentally for at least some polymers. The boundaries of the universal K³ region and the plateau region are determined theoretically here using precise quantitative criteria for universal or plateau behavior of D_app(K) for a Rouse-Zimm model containing N + 1 subchains with rms subchain extension b. Allowing a maximum of 13% nonuniversal behavior, the domain of the universal K³ region is given by K²R²_G = K²Nb²/6 ≥ 7 and K²b² ? 0.54. Allowing as much as 10% nonplateau behavior, the boundary for onset of plateau behavior is K²b² = 18.3. D_app(K) is at least 50% nonuniversal when K²b²/6 = 6 ln 3. Extension of these results to DNA is examined theoretically, and good agreement of the pertinent predictions with published experimental data is demonstrated. It is concluded that no truly universal K³ region exists for DNA with M_r ? 10⁷ and persistence length a ≥ 450 Å, although marginally (?17% nonuniversal) universal behavior, is exhibited in a very narrow domain 0.64 × 10¹⁰ ? K² ? 0.84 × 10¹⁰ cm^?2 for ?29 DNA (M_r = 11.5 × 10⁶). More than 50% of D_app(K) is governed by local (elastic) rigid-body motions when K² = 5.23 × 10¹⁰ cm^?2. The existence of a very wide region of nonuniversal apparent K³ behavior extending up to very large K², far into the plateau region, is demonstrated in a plot of D_app(K)/K vs K² for the Rouse-Zimm model. This is shown to stem in part from visual artifacts of plotting D_app(K)/K vs K², even for rigid species.     

Effect of Water Stress and Mercuric Chloride on the Translational Diffusion of Water in Maize Seedling Roots

Water diffusion in maize roots (Zea mays L., cv. Donskaya 1) was investigated with a pulsed gradient NMR using mercuric chloride as an inhibitor of water channels in cell membranes. A novel operation program was applied that allowed selective evaluation of fractional amounts of water transported through various pathways—the apoplastic, symplasmic, and transmembrane routes. The blockage of water channels with HgCl₂ reduced the rates of water diffusion by a factor of 1.5–2. This effect was reversible and was removed by the addition of -mercaptoethanol. The coefficient of water diffusion changed with time elapsed after the HgCl₂ treatment. The effect of water stress on the rates of water diffusion was similar to that of HgCl₂. Remarkably, the water-stressed roots of maize seedlings were insensitive to the inhibitor of water channels. The results are interpreted in terms of redistribution of water flows among various routes in plant tissues. Water stress and mercuric chloride treatments decelerate the transmembrane water transport and promote water flow along the apoplastic pathway. These responses might arise from the reversible regulation of water movement along various transport pathways.     

Intramolecular interference effects in dynamic light scattering from rigid rings

A formula for the apparent diffusion coefficient [D_app(κ)] of a rigid ring is derived from the exact first cumulant of its dynamic structure factor. D_app(κ) is expressed in terms of the ring radius, the diffusion coefficients (D_zz and D_xx) for translation parallel and perpendicular, respectively, to the symmetry axis, and the diffusion coefficients (D and D) for rotation around the symmetry and transverse axes, respectively. D_app(κ) exhibits oscillations as a function of the scattering vector k , which depend on D and the anisotropy of translational diffusion (D_zz ? D_xx). The maxima in D_app(κ) are associated with minima in the static structure factor S(κ, 0), which are due to destructive intramolecular interference. The oscillations in D_app(κ) result from periodic variations in the relative intensities of scattered light from different orientations of the ring, which manifest the various motions to different extents. The orientations contributing most to the scattered intensity are those that exhibit the least destructive interference and consequently contribute most to the decay of the dynamic structure factor. © 1993 John Wiley & Sons, Inc.     

Regulation of Intercellular Water Exchange in Various Zones of Maize Root under Stresses

Apical root meristems and segments of root elongation zone were sampled from 4- to 5-day-old Zea mays L. seedlings. The vacuolar ATPase and pyrophosphatase, the tonoplast marker enzymes, and the tonoplast -, -, and -aquaporins were visualized by means of indirect immunofluorescent microscopy with the use of the respective antibodies. Following cell plasmolysis (700 mM mannitol, 2.5 h), the vacuolar ATPase and pyrophosphatase were detected in cell wall pores where plasmodesmata remained detached from the plasmolyzed protoplasts. This finding provides further evidence for existence of the vacuolar symplast in the elongation zone of maize root, which may ensure intercellular continuity of plant tissues. The pulsed NMR method was used to study the self-diffusion of water molecules. The diffusive decay in the root elongation zone was nonexponential, and it was transformed to three exponential terms with characteristic coefficients of self-diffusion; two of these coefficients (D ₂ and D ₃) characterize the water self-diffusion in the cytoplasmic and vacuolar symplasts of root, respectively. The root apical meristem was also investigated with NMR technique by virtue of paramagnetic doping of the apoplast. This approach allowed selective studying of water diffusion within the symplast compartments. Partial dehydration with PEG-6000, 12 and 20%, for 2.5 h and chemical stressors (ABA and salicylic acid, 0.1 mM, 24 h) were applied to modify water permeability of plasmodesmata and tonoplast aquaporins. The transcellular water permeability increased in the root meristem under the action of all stress factors. In the root elongation zone exposed to partial dehydration, the water exchange in the apoplast became the dominant component. Other stress factors affected water relations in different manners. ABA elevated the water permeability of the vacuolar symplast, in contrast to salicylic acid that decreased water conductance of both the cytoplasmic and vacuolar symplasts.