Low-temperature Signal Research Articles

Low-temperature signal transduction: induction of cold acclimation-specific genes of alfalfa by calcium at 25 degrees C.

To study the role of calcium in cold acclimation, we examined the relationship between calcium influx and accumulation of transcripts of two cas (cold acclimation-specific) genes of alfalfa, cas15 and cas18. Whereas a decline in temperature from 25 to 15 degrees C had little effect on the influx of extracellular 45Ca2+, an increasing influx was observed when the temperature was lowered further. The influx of 45Ca2+ at 4 degrees C was nearly 15 times greater than at 25 degrees C. The addition of calcium chelators or of calcium channel blockers, which have been shown to prevent cold acclimation, inhibited the influx of extracellular 45Ca2+ as well as the expression of cas genes at 4 degrees C. The addition of a calcium ionophore or a calcium channel agonist to nonacclimated cells caused the influx of extracellular 45Ca2+ and induced the expression of cas genes at 25 degrees C. These results suggest that a cold-induced calcium influx plays an essential role in cold acclimation. To further study the role of calcium, we isolated two sequences corresponding to calcium-dependent protein kinases. The transcript level of one of them was markedly upregulated at 4 degrees C. We propose a sequence of signaling events that is likely to occur early during cold acclimation and leads to the expression of cas genes and the development of freezing tolerance.

Low-Temperature Signal Transduction: Induction of Cold Acclimation-Specific Genes of Alfalfa by Calcium at 25 degrees C

Low-Temperature Signal Transduction: Induction of Cold Acclimation-Specific Genes of Alfalfa by Calcium at 25 degrees C

Investigation of the magnetic field-induced microwave absorption across Tc in single crystals of YBa 2Cu 3O 7−δ and BiSrCaCuO : Anisotropy and magnetic field dependence

The magnetic field-modulated microwave absorption near T c has been studied in high-quality single crystals of YBa 2Cu 3O 7− x ( T c ≈ 90 K) and in BiSrCaCuO ( T c ≈ 85 K) as a function of magnetic field B < 1.3 T and orientation. Sharp microwave absorption peaks were observed when sweeping the temperature through T c from above. The analysis of the peaks using Tinkham's model for fluxon losses gives values for the pinning strength of A ≈ 1000 T and A ≈ 3 T for the YBaCuO and BSCCO crystals, respectively. The anisotropy values obtained from the orientation dependence of the peaks were ϵ = 5 and ϵ = 40−60 for these two compounds, respectively. The results are also discussed with respect to the B-T vortex phase diagram. A marked difference between the YBaCuO and BSCCO compounds is the presence of a low-temperature absorption signal with a different orientation dependence, observed only in the BSCCO compound, in addition to the signals just below T c observed in both compounds.

Multifrequency EPR investigations into the origin of the S2-state signal at g = 4 of the O2-evolving complex

The low-temperature S2-state EPR signal at g = 4 from the oxygen-evolving complex (OEC) of spinach Photosystem-II-enriched membranes is examined at three frequencies, 4 GHz (S-band), 9 GHz (X-band) and 16 GHz (P-band). While no hyperfine structure is observed at 4 GHz, the signal shows little narrowing and may mask underlying hyperline structure. At 16 GHz, the signal shows g-anisotropy and a shift in g-components. The middle Kramers doublet of a near rhombic S = 12 system is found to be the only possible origin consistent with the frequency dependence of the signal. Computer simulations incorporating underlying hyperfine structure from an Mn monomer or dimer are employed to characterize the system. The low zero field splitting (ZFS) of D = 0.43 cm and near rhombocity of E/D = 0.25 lead to the observed X-band g value of 4.1. Treatment with F or NH3, which compete with Cl for a binding site, increases the ZFS and rhombicity of the signal. These results indicate that the origin of the OEC signal at g = 4 is either an Mn(II) monomer or a coupled Mn multimer. The likelihood of a multimer is favored over that of a monomer.

Multifrequency EPR investigations into the origin of the S2-state signal at g = 4 of the O2-evolving complex

The low-temperature S2-state EPR signal at g = 4 from the oxygen-evolving complex (OEC) of spinach Photosystem-II-enriched membranes is examined at three frequencies, 4 GHz (S-band), 9 GHz (X-band) and 16 GHz (P-band). While no hyperfine structure is observed at 4 GHz, the signal shows little narrowing and may mask underlying hyperline structure. At 16 GHz, the signal shows g-anisotropy and a shift in g-components. The middle Kramers doublet of a near rhombic S = 12 system is found to be the only possible origin consistent with the frequency dependence of the signal. Computer simulations incorporating underlying hyperfine structure from an Mn monomer or dimer are employed to characterize the system. The low zero field splitting (ZFS) of D = 0.43 cm and near rhombocity of E/D = 0.25 lead to the observed X-band g value of 4.1. Treatment with F or NH3, which compete with Cl for a binding site, increases the ZFS and rhombicity of the signal. These results indicate that the origin of the OEC signal at g = 4 is either an Mn(II) monomer or a coupled Mn multimer. The likelihood of a multimer is favored over that of a monomer.

Directed mutagenesis of an iron-sulfur protein of the photosystem I complex in the filamentous cyanobacterium Anabaena variabilis ATCC 29413.

In oxygenic photosynthetic organisms the PSI-C polypeptide, encoded by the psaC gene, provides the ligands for two [4Fe-4S] centers, FA and FB, the terminal electron acceptors in the photosystem I (PSI) complex. An insertion mutation introduced in the psaC locus of the filamentous cyanobacterium Anabaena variabilis ATCC 29413 resulted in the creation of a mutant strain, T398-1, that lacks the PSI-C polypeptide. In medium supplemented with 5 mM fructose, the mutant cells grew well in the dark. However, when grown in the same medium under light, the doubling rate of T398-1 cells was significantly decreased. In intact cells of T398-1, bicarbonate-dependent whole-chain electron transport (PSII and PSI) could not be detected, although partial electron transport reactions involving either one of the two photosystems could be measured at significant rates. The low-temperature EPR signals attributed to the [4Fe-4S] centers FA and FB were absent in the mutant cells. Chemical titration measurements indicated that the ratios of chlorophyll to the primary donor P700 were virtually identical in membranes from the wild-type and mutant cells. Moreover, room-temperature optical spectroscopic analysis of the thylakoid membranes isolated from T398-1 showed flash-induced P700 oxidation followed by dark rereduction, indicating primary photochemistry in PSI. Thus stable assembly of the reaction center of PSI can occur in the absence of the Fe-S cluster cofactors FA and FB. These studies demonstrate that Anabaena 29413 offers a useful genetic system for targeted mutagenesis of the PSI complex.

Travelling wave drift mobility measurements of photoexcited carriers in a-Si:H at low temperatures

Abstract The travelling wave technique was used to measure the drift mobility of photoexcited carriers in a-Si:H from 1·6 to 300 K. Above 100 K the drift mobility increases with temperature as expected from multiple trapping theories. Below 100K the measured signal increases with decreasing temperature reaching values that, if interpreted as drift mobility, exceed 07middot;2 cm2 V−1s−1 at 1·6 K. The results are qualitatively consistent with the large drift mobilities measured at low temperatures by the time-of-flight technique. The drift mobility increases linearly with travelling wave frequency below l00 K but is independent of frequency above 100 K. However, the interpretation of the low-temperature signal as drift mobility is in doubt because the sign of the signal below 100 K, which should indicate the sign of the dominant carrier, is instead determined by the orientation of the y axis of the LiNbO3 plate used to generate the travelling wave. This indicates a heretofore unknown interaction of the trav...

Zinc diffusion in GaAs and zinc-induced disordering of GaAs/AlGaAs multiple quantum wells: a multitechnique study

A review of experimental results obtained by different techniques is presented on the problem of zinc diffusion. Zinc diffusion was carried out on Si-doped GaAs (n almost-equal-to 10(18) cm-3) and on multiple quantum well (MQW) structures. The samples were investigated by secondary-ion mass spectroscopy (SIMS), different imaging modes of scanning electron microscopy such as secondary electrons, cathodoluminescence (CL) and electron beam-induced current (EBIC), transmission electron microscopy on a wedge-shaped specimen (WTEM) and by photoluminescence (PL). A nonexponential decay of the low-temperature EBIC signal accompanied by a very low CL signal due to the high density of nonradiative recombination centres were observed in the diffused region of the n-doped GaAs. Indeed, PL measurements demonstrate that Ga vacancies play a key role on the mechanism of the Zn diffusion. On the impurity-induced disordered (IID) MQW samples, an enrichment of Al at the surface was observed by SIMS and confirmed by WTEM and PL. Low-temperature PL spectra show the gradual disappearance of the MQW excitonic transitions as the number of disordered layers increases. When all of the MQW structure is destroyed, the band-to-band recombinations in the IID produced alloy dominate the PL spectrum.

Growth and properties of oxygen- and ion-doped Bi2Sr2CaCu2O8+ delta single crystals.

A directional solidification method for growing large single crystals in the ${\mathrm{Bi}}_{2}$${\mathrm{Sr}}_{2}$${\mathrm{CaCu}}_{2}$${\mathrm{O}}_{8+\mathrm{\ensuremath{\delta}}}$ system is reported. Ion doping, with replacement of La for Sr and Y for Ca, as well as oxygen doping in these crystals has been explored. Doped and undoped crystals have been characterized using microprobe analysis, x-ray diffraction, thermogravimetric analysis, and magnetic and Hall measurements. Ion doping results in little change of the superconducting transition for substitution levels below 20--25%, while beyond this level the Meissner signal broadens and the low-temperature Meissner signal decreases. Microprobe analysis and x-ray diffraction performed on these more highly substituted single crystals provide evidence for inhomogeneity and phase segregation into regions of distinct composition. Annealing unsubstituted crystals in increasing partial pressures of oxygen reversibly depresses the superconducting transition temperature from 90 (as made) to 77 K (oxygen pressure annealed), while the carrier concentrations, as determined from Hall effect measurements, increase from n=3.1(3)\ifmmode\times\else\texttimes\fi{}${10}^{21}$ ${\mathrm{cm}}^{\mathrm{\ensuremath{-}}3}$ (0.34 holes per Cu site) to 4.6(3)\ifmmode\times\else\texttimes\fi{}${10}^{21}$ ${\mathrm{cm}}^{\mathrm{\ensuremath{-}}3}$ (0.50 holes per Cu site).No degradation of the Meissner transition or other indications of inhomogeneity or phase segregation with doping are noted, suggesting that oxygen-doped ${\mathrm{Bi}}_{2}$${\mathrm{Sr}}_{2}$${\mathrm{CaCu}}_{2}$${\mathrm{O}}_{8+\mathrm{\ensuremath{\delta}}}$ is a suitable system for pursuing doping studies. The decrease in ${\mathit{T}}_{\mathit{c}}$ with concentration for 0.34\ensuremath{\le}n\ensuremath{\le}0.50 indicates that a high-carrier-concentration regime exists in which ${\mathit{T}}_{\mathit{c}}$ decreases with n and suggests that this decrease does not arise from material inhomogeneity or other materials problems. An examination of the variation of ${\mathit{T}}_{\mathit{c}}$ with the density of states and lattice constants for all of the doped and undoped superconducting samples considered here indicates that changes in ${\mathit{T}}_{\mathit{c}}$ with doping are primarily affected by changes in the density of states (or carrier concentration) rather than by structural variation induced by the doping.

Ubisemiquinone in the NADH-ubiquinone reductase region of the mitochondrial respiratory chain

Coupled bovine heart submitochondrial particles exhibit a rotenone-sensitive g = 2.00 low-temperature EPR signal attributable to ubisemiquinone which is observed during steady-state electron transfer from NADH to oxygen or from succinate to NAD + in Δ \\ ̃ gmH +-dependent reverse electron transfer. Quantitation of the signal under optimal conditions gives a value for maximal semiquinone of approx. 0.5 spins per spin of the fully reduced NADH dehydrogenase iron-sulfur center N-2. The intensity of the signal is drastically reduced when electron transfer from NADH to oxygen is blocked by cyanide or in the case of the reverse electron transfer from succinate to NAD + being prevented by anaerobiosis. Only those particles which contain ‘turnover-preconditioned” NADH-ubiquinone reductase demonstrate the ubisemiquinone signal together with N-1, N-2, N-3 and N-4 iron-sulfur centers. The spin relaxation characteristics of the rotenone-sensitive ubisemiquinone signal point to its interaction with one of the rapidly relaxing (4Fe-4S) centers, most probably N-2.

Precautions when interpreting EPR and dc magnetization measurements of high-Tc RBa2Cu3Oy-phase superconducting materials.

Many electron-paramagnetic-resonance (EPR) and magnetic susceptibility measurements for the high-${T}_{c}$ superconductors of the form $R{\mathrm{Ba}}_{2}{\mathrm{Cu}}_{3}{\mathrm{O}}_{9\ensuremath{-}x}$ ($R=\mathrm{Y}$ or a rare earth) have been reported. Excluding local moment resonances due solely to the $R$ atoms, we show that all of the EPR measurements known to us are not intrinsic to the superconducting phase, but rather are due to low concentrations of spurious phases typically present in the samples. BaCu${\mathrm{O}}_{2}$ is found to be the main source of the low-temperature EPR signal and of the magnetic susceptibility which has been reported for $\mathrm{Y}{\mathrm{Ba}}_{2}{\mathrm{Cu}}_{3}{\mathrm{O}}_{9\ensuremath{-}x}$ above ${T}_{c}$.

The state of manganese in the photosynthetic apparatus: 5. The chloride effect in photosynthetic oxygen evolution. Is halide coordinated to the EPR-active manganese in the O 2-evolving complex? Studies of the substructure of the low-temperature multiline EPR signal

The role of chloride in the manganese-containing oxygen-evolving complex of Photosystem II has been studied by observing the amplitude of the multiline EPR signal as a function of Cl − concentration or when Cl − is replaced by Br − or F −. The correlation of the multiline EPR signal intensity and O 2 activity with the concentration of Cl − shows that chloride is involved in oxygen evolution at the S 2 or earlier S states, and that it is necessary for the production of an EPR-detectable S 2 state. We have developed a new method for the preparation of subchloroplast PS II particles containing Br − and F −) and have used these particles for studying the EPR fine structure at high resolution. The fine structure shows a multiplet of 4–6 lines with 10–15 G spacing; at the resolution of our experiment there are no significant differences between the Cl −-and Br −-containing samples, suggesting that the halide is not a ligand of the EPR-active Mn. Various structural possibilities for the Mn complex, which would account for the observed fine structure of the multiline EPR spectrum are discussed.

Roles of three lumen-surface proteins in the formation of S 2 state and O 2 evolution in Photosystem II particles from spinach thylakoid membranes

Reconstitution of O 2 evolution in CaCl 2-treated PS II at the level of the original PS II was achieved by simultaneous reinsertion of the three lumen-surface proteins, 17, 23 and 34 kDa proteins, and total thylakoid lipids in the presence of 25% glycerol and 15 mM sodium cholate. By means of this reconstitution method, the following was found, concerning the roles of the three proteins in the formation of the S 2 state and O 2 evolution of the water-splitting complex. (1) Complete removal of the three proteins from the PS II particles by 1 M CaCl 2 treatment caused a rapid release of Mn from the particles when incubated at 25°C, but not at 0°C. The rapid release of Mn at 25°C was prevented by the reinsertion of the 34 kDa protein but not by the other two proteins. (2) O 2 evolution was recovered in CaCl 2-treated PS II by reinserting the 34 kDa protein, when Cl − was present at a level of 50 mM, but it declined when the Cl − concentration was decreased. Upon further addition of the 23 kDa protein, O 2-evolving activity was enhanced to the original PS II level at concentrations of Cl − higher than 10 mM. Below this concentration, besides the 23 and 34 kDa proteins, the 17 kDa protein was required for maximal O 2 evolution. (3) The effects of the three proteins and Cl − on the recovery of the low-temperature multiline EPR signal in the CaCl 2-treated PS II were essentially the same as those on the recovery of the O 2 evolution, indicating that the three proteins and Cl − contribute to the advancement of the water-splitting complex to the S 2 state.

NADH and NADPH dehydrogenases from the blue-green alga, Aphanocapsa

Two different NAD(P)H dehydrogenases could be demonstrated in the blue-green alga, Aphanocapsa. Both function as quinone reductases using benzoquinone as electron acceptor. One, which was found in the soluble fraction, was NADH specific and showed high sensitivity to rotenone, thenoyltrifluoroacetone and o-phenanthroline. The second dehydrogenase was membrane-bound and used NADH as well as NADPH as substrates. Inhibition by rotenone and o-phenanthroline was less pronounced with the bound enzyme than with the soluble enzyme. Based on studies with NADH or NADPH, the membrane-bound enzyme apparently was associated with a low-temperature EPR signal at g=1.92 in the reduced state, indicative of an iron-sulfur center. The membrane-bound dehydrogenase was solubilized with Triton X-100 and partially purified. This preparation was used for studies of enzyme kinetics and acceptor specificity.

Physiochemical properties of ferralterin, a regulatory iron-sulfur protein functional in oxygenic photosynthesis

Ferralterin, an iron-sulfur protein identified earlier in chloroplasts and cyanobacteria, was purified to homogeneity from spinach leaves and Nostoc muscorum cells. When isolated from both sources, ferralterin showed a molecular weight of about 28,000 and was comprised of three subunits: one of molecular weight 12,000 and two, apparently identical, of molecular weight 7000. Based on the Lowry method of protein estimation, ferralterin contained approximately 3 g atoms each of nonheme iron and acid-labile sulfide per mole. The iron-sulfur cluster of ferralterin showed unusual redox and electron paramagnetic resonance (EPR) properties. Ferralterin was EPR silent as isolated and did not show an EPR signal on addition of reductants such as sodium dithionite or on exposure to illuminated chloroplast membranes. These reducing conditions also had no significant effect on the absorption spectrum of isolated ferralterin. The ferralterin iron-sulfur cluster was oxidized selectively by ferricyanide and showed a midpoint redox potential of +410 mV. Ferricyanide-oxidized ferralterin was characterized by a low-temperature EPR signal with g values of 2.10, 2.05, and 2.00 (spinach) and 2.09, 2.04, and 1.98 ( Nostoc). When oxidized by ferricyanide, the iron-sulfur cluster could be reduced by a variety of reductants, including illuminated chloroplast membranes. The results are consistent with the conclusion that, like several other iron-sulfur enzymes (aconitase, glutamine phospho-ribosylpyrophosphate amidotransferase, hydrogenase), ferralterin achieves its catalytic effect via an active group independently of a redox change in the iron-sulfur chromophore.

ChemInform Abstract: 13C‐NMR SPECTROSCOPIC AND CONFORMATIONAL STUDIES ON ROTATIONS IN SUBSTITUTED ETHANES

Possibilities and results of 13C-DMR line shape analysis are explored with 1X-2,2-dimethylpropanes (neopentanes, 1), 2X-3,3-dimethylbutanes (2) and 2X-2,3,3-trimethylbutanes (3) with X = H, Hal, OR or CH3. The observed barriers ΔG* consistently increase in this sequence, e.g. for X = Cl from 6.5 (1) to 7.7 (2) to 10.4 (3) kcal/mol. Molecular mechanics force field calculations for 1, although giving barriers too low by 0.5–1.5 kcal/mol, correctly reproduce the observed trends and explain the puzzling low barriers observed for X = CH3 in comparison to X = Hal, as the result of the higher ground state energy of the methyl compounds. Conformational equilibria are investigated with the non-topomeric 2X-2,3-dimethylbutanes. The ΔG0 values obtained from low temperature signal integration agree with force field predictions only for X = H and F, which dominate, as X = OR does, in the all-gauche conformation; in contrast, the predominance of the trans conformer for X = Cl, Br and J is not rationalized. The calculated ground state geometries show no significant geometry distortion upon change of the substituents X, which provides a basis for the 13C shielding comparison to corresponding cyclohexanes of similar substitution pattern. The observed substituent effects on the 13C shifts are in agreement with other models and mechanistic proposals for the screening constant variations.

The temperature dependence of 13C NMR shifts in polar compounds and its role for the determination of conformational equilibria

AbstractIntrinsic temperature dependencies of 13C NMR shifts in alkanes bearing one polar C‐αX bond are determined with neopentyl and 4‐tert‐butylcyclohexyl derivatives as conformationally homogeneous model compounds. The increased shiedling for C‐α at higher temperatures can be related to a C‐α—X bond length increase and, for less polarizable CX bonds, essentially to a decrease of solvent polarity on raising the temperature. The use of temperature dependent 13C shifts in conformationally mixed compounds for the determination of the equilibrium constants, K, is evaluated with n‐propyl halides; the computer fit of the unknown conformer shifts and the conformational enthalphy difference, δH° to the time averaged shifts yields δH° values which, although converging rather broadly, are in general agreement with literature data. In compounds with higher conformational barriers, such as methoxy‐ and bromocyclohexane, low temperature signal integration yields accurate δG° values; inclusion of shifts above coalescence, however, yields unreliable δH° and δS° parameters. This can only partially be remedied by application of temperature shift corrections obtained from parent t‐butylcyclohexyl compounds.

13C‐NMR Spectroscopic and Conformational Studies on Rotations in Substituted Ethanes

AbstractPossibilities and results of 13C‐DMR line shape analysis are explored with 1X‐2,2‐dimethylpropanes (neopentanes, 1), 2X‐3,3‐dimethylbutanes (2) and 2X‐2,3,3‐trimethylbutanes (3) with X = H, Hal, OR or CH3. The observed barriers ΔG* consistently increase in this sequence, e.g. for X = Cl from 6.5 (1) to 7.7 (2) to 10.4 (3) kcal/mol. Molecular mechanics force field calculations for 1, although giving barriers too low by 0.5–1.5 kcal/mol, correctly reproduce the observed trends and explain the puzzling low barriers observed for X = CH3 in comparison to X = Hal, as the result of the higher ground state energy of the methyl compounds. Conformational equilibria are investigated with the non‐topomeric 2X‐2,3‐dimethylbutanes. The ΔG0 values obtained from low temperature signal integration agree with force field predictions only for X = H and F, which dominate, as X = OR does, in the all‐gauche conformation; in contrast, the predominance of the trans conformer for X = Cl, Br and J is not rationalized. The calculated ground state geometries show no significant geometry distortion upon change of the substituents X, which provides a basis for the 13C shielding comparison to corresponding cyclohexanes of similar substitution pattern. The observed substituent effects on the 13C shifts are in agreement with other models and mechanistic proposals for the screening constant variations.

The Effect of Light at Different Wavelengths on Electron Spin Resonances in Wool

An action spectrum from 280 nm to 390 nm for the formation of a non-sulphur-free radical in wool at room temperature and in the absence of air has been determined. This spectrum shows a peak at 285 nm, indicating that the aromatic amino acid components of keratin are important chromophores in the formation of these radicals. A low-temperature ESR signal in wool at g ~ 4.25 decreases on irradiation with light from a mercury arc through a Wratten 35 filter (320–470 nm). This signal arises from a ferric ion—keratin complex, and the photodegradation of this complex is accompanied by the formation of non-sulphur type radicals at room temperature.

Low-Temperature Signal Detection in Optically Pumped Rubidium Vapor

Low-Temperature Signal Detection in Optically Pumped Rubidium Vapor