Effect of cold acclimation on bulk tissue electrical impedance: I. Measurements with birdsfoot trefoil at subfreezing temperatures

The resistive and reactive components of electrical impedance were measured for birdsfoot trefoil (Lotus corniculatus L.) stems at freezing temperatures to −8°C. As temperature decreased the specific resistance at frequencies between 49 hertz and 1.11 megahertz of stems from cold acclimated plants increased more rapidly than from nonacclimated plants. This temperature dependence of specific resistance could be characterized by an Arrhenius activation energy; cold acclimated stems had a larger Arrhenius activation energy than nonacclimated stems. The low frequency resistance is believed to characterize the extracellular region of the stems and the high frequency resistance is believed to characterize the intracellular region of the stems. Cold acclimation increased the intracellular but not the extracellular resistance at nonfreezing temperatures. Cold acclimated stems were not injured by freezing to −8°C and thawing, but nonacclimated stems were injured by freezing to temperatures between −2.2 and −5.6°C and thawing. Injury to nonacclimated stems at freezing temperatures below −2.2°C was indicated by a decrease in the ratio of resistance at 49 Hz to that at 1.11 megahertz.     

In vivo plant impedance measurements and characterization of membrane electrical properties: The influence of cold acclimation

A procedure to measure both the real (resistance) and complex (reactance) part of plant tissue impedance is described. Bare metal electrodes were used and the impedance of electrodes was determined by obtaining measurements at four interelectrode distances. The frequency (f) dependence of the impedance of birdsfoot trefoil (Lotus corniculatus L.) stems could be modeled, approximately, by a resistor representing an extracellular resistance, a resistor representing an intracellular resistance, and a resistor and capacitor representing cell membranes. However, the membrane parameters appeared to be frequency dependent. Therefore, they were characterized by calculating a time constant at each frequency. For example, for one plant stem the time constant (τ) decreased from 1.19 × 10⁻³ sec at 101 hz to 1.36 × 10⁻⁶ sec at 100 KHz. This decrease with frequency could be described by an equation of the form: ln(τ) = a + bln(f) + c(ln[f])². Cold acclimation increased (P ⩽ 0.05) the intracellular resistance. But cold acclimation did not have a significant effect on estimates of the extracellular resistance, the membrane resistance, or the membrane time constant. Depending on the cultivar, cold acclimation either decreased or increased the estimate of membrane capacitance.     

Behavior and viability of tobacco protoplasts in response to electrofusion parameters

This investigation examines responses of protoplasts in a systematic and quantitative way to the various electrical treatments used to achieve electrofusion and their individual and cumulative effect on protoplast viability. Mesophyll and cell suspension protoplasts from two species of the same genera, Nicotiana tabacum and N. rustica var brasilia were used in these experiments. Optimal frequencies for alignment of tobacco protoplasts were between 500 kilohertz and 2 megahertz at 100 volts per centimeter. Variations in frequency and voltage of the alternating current (AC) field caused predictable movements of protoplasts within an electrofusion chamber. AC frequencies below 10 hertz or above 5 megahertz significantly decreased the viability of protoplasts in the fusion chamber as estimated by fluorescein diacetate staining 1 hour after treatment. Although the direct current (DC) pulse appeared to have a slight detrimental effect on protoplast viability, this effect was not significantly different from untreated control preparations.

Protoplasts from both leaf mesophyll cells and suspension cells were induced to fuse with one or more 10 to 30 microseconds DC square wave pulses of approximately 1 kilovolt per centimeter after the protoplasts had been closely appressed with an AC field.

     

Relationship of Electrical Conductance at Two Frequencies to Cold Injury and Acclimation in Cornus stolonifera Michx

The ratio of electrical conductance measured at two frequencies can be used to predict the cold hardiness of stem sections of Cornus stolonifera Michx. during the first stage of cold acclimation. Electrical conductance at 50 hertz divided by electrical conductance at 100 kilohertz gave a better estimate of hardiness than measurements at either frequency alone. The observed increase in the electrical conductance ratio as hardiness increased is consistent with an increase in membrane permeability. After plants were exposed to nonlethal frost, hardiness increased rapidly, and the relation between the conductance ratio and hardiness changed. This change indicates that ice crystallization induces a significant physiological alteration in the plants. Contrary to expectations, stem sections exposed to lethal temperatures could not consistently be separated from sections exposed to nonlethal temperatures by electrical conductance ratio measurements made immediately after thawing.     

Rapid Assessment of the Physiological Status of the Polychlorinated Biphenyl Degrader Comamonas testosteroni TK102 by Flow Cytometry

The viability of the polychlorinated biphenyl-degrading bacterium Comamonas testosteroni TK102 was assessed by flow cytometry (FCM) with the fluorogenic ester Calcein-AM (CAM) and the nucleic acid dye propidium iodide (PI). CAM stained live cells, whereas PI stained dead cells. When double staining with CAM and PI was performed, three physiological states, i.e., live (calcein positive, PI negative), dead (calcein negative, PI positive), and permeabilized (calcein positive, PI positive), were detected. To evaluate the reliability of this double-staining method, suspensions of live and dead cells were mixed in various proportions and analyzed by FCM. The proportion of dead cells measured by FCM directly correlated with the proportion of dead cells in the sample (y = 0.9872 x + 0.18; R² = 0.9971). In addition, the proportion of live cells measured by FCM inversely correlated with the proportion of dead cells in the sample (y = −0.9776 x + 98.36; R² = 0.9962). The proportion of permeabilized cells was consistently less than 2%. These results indicate that FCM in combination with CAM and PI staining is rapid (≤1 h) and distinguishes correctly among live, dead, and permeabilized cells.     

Differentiation of isomeric Lewis blood groups by positive ion electrospray tandem mass spectrometry

Lewis histo-blood group antigens are one of the major classes of biologically active oligosaccharides. In this work, underivatized Lewis blood groups were studied by electrospray tandem mass spectrometry (ESI-MSⁿ) in the positive mode with three different mass analyzers: Q-TOF (quadrupole time-of-flight), QqQ (triple quadrupole), and LIT (linear ion trap). It was observed that, under collision-induced fragmentations, type 1 Lewis antigens (Le^a and Le^b) could be distinguished from type 2 (Le^x and Le^y) on the basis of specific fragmentations of the GlcNAc unit. Whereas O-4-linked sugars of the GlcNAc are lost as residues, the O-3-linked sugars undergo fragmentation both as sugar units and as sugar residues (unit −18 Da). Type 2 Lewis antigens also showed a characteristic cross-ring cleavage ^0,2A₂ of the GlcNAc. As a result, the product ions at m/z 388 and 305, characteristic of Le^x, and m/z 372, characteristic of Le^a, are proposed to distinguish the trisaccharide isomers Le^x/Le^a. Also, the product ions at m/z 534 and 305, characteristic of Le^y, and m/z 372, characteristic of Le^b, are proposed to distinguish the tetrasaccharide isomers Le^b/Le^y. These diagnostic fragment ions were further applied in the identification of Lewis type 2 antigens (Le^x and Le^y) in the lipopolysaccharide of the human gastric pathogen, Helicobacter pylori.     

Is There a Link between the Lipopolysaccharide of Helicobacter pylori Gastric MALT Lymphoma Associated Strains and Lymphoma Pathogenesis?

The aim of this study was to investigate the Lewis antigen expression in Helicobacter pylori gastric MALT lymphoma associated strains in comparison to chronic gastritis only strains. Forty MALT strains (19 cagPAI (−) and 21 cagPAI (+)) and 39 cagPAI frequency-matched gastritis strains (17 cagPAI (−) and 22 cagPAI (+)) were included in this study. The lipopolyssacharide for each strain was extracted using a hot phenol method and the expression of Le^x and Le^y were investigated using Western Blot. The data were analyzed according to the strains'' cagPAI status and vacA genotype. Le^x was identified in 21 (52.5%) MALT strains and 29 (74.3%) gastritis strains. Le^y was identified in 30 (75%) MALT strains and 31 (79.5%) gastritis strains. There was an association between cagPAI positivity and Le^x expression among MALT strains (p<0.0001), but not in gastritis strains (p = 0.64). Among cagPAI (−) strains, isolates expressing solely Le^y were associated with MALT with an odds ratio of 64.2 (95% CI 4.9–841.0) when compared to strains expressing both Le^x and Le^y. vacA genotypes did not modify the association between Lewis antigen expression and disease status. In conclusion, cagPAI (−) MALT strains have a particular Lewis antigen profile which could represent an adaptive mechanism to the host response or participate in MALT lymphomagenesis.     

Chill-tolerant Gryllus crickets maintain ion balance at low temperatures

Insect cold tolerance is both phenotypically-plastic and evolutionarily labile, but the mechanisms underlying this variation are uncertain. Chill-susceptible insects lose ion and water homeostasis in the cold, which contributes to the development of injuries and eventually death. We thus hypothesized that more cold-tolerant insects will better maintain ion and water balance at low temperatures. We used rapid cold-hardening (RCH) and cold acclimation to improve cold tolerance of male Gryllus pennsylvanicus, and also compared this species to its cold-tolerant relative (Gryllus veletis). Cold acclimation and RCH decreased the critical thermal minimum (CT_min) and chill coma recovery time (CCR) in G. pennsylvanicus, but while cold acclimation improved survival of 0 °C, RCH did not; G. veletis was consistently more cold-tolerant (and had lower CCR and CT_min) than G. pennsylvanicus. During cold exposure, hemolymph water and Na⁺ migrated to the gut of warm-acclimated G. pennsylvanicus, which increased hemolymph [K⁺] and decreased muscle K⁺ equilibrium potentials. By contrast, cold-acclimated G. pennsylvanicus suffered a smaller loss of ion and water homeostasis during cold exposure, and this redistribution did not occur at all in cold-exposed G. veletis. The loss of ion and water balance was similar between RCH and warm-acclimated G. pennsylvanicus, suggesting that different mechanisms underlie decreased CCR and CT_min compared to increased survival at 0 °C. We conclude that increased tolerance of chilling is associated with improved maintenance of ion and water homeostasis in the cold, and that this is consistent for both phenotypic plasticity and evolved cold tolerance.     

EPR,ligand field spectra and antimicrobial activity of dimeric aryloxyacetatocopper(II) complexes with antipyrine

Copper(lI) complexes with aryloxyacetato ligands and antipyrine (Apy) of the general formula [Cu₂- (RCOO)₄(Apy)₂] were prepared with RCOO⁻ as phenoxyacetato, 3-methylphenoxyacetato, 4-methylphenoxyacetato, 4-chlorophenoxyacetato, 2-methyl- 4-chlorophenoxyacetato, 3-methyl-4-chlorophenoxyacetato and 2-naphthoxyacetato anions. The parameters of the triplet spin state EPR spectra at X and Q band frequencies (D|hc = 0.37 cm⁻¹, g⊥ = 2.07, g6 = 2.38, A6|c = 0.007 cm⁻¹) provide evidence of a dimeric carboxylato-bridged structure with apically bound antipyrine. The almost constant EPR data as well as the energies of the ligand field transitions at 77 K [d_x²−y² ← d_xz, d_yz (14000 cm⁻¹), d_x²−y² ← d_xy (11200 cm⁻¹) and d_x²−y² ← d_z² (9500 cm⁻¹)] indicate that the square-pyramidal geometry of the CuO₅ polyhedra is nearly unaffected by the substituents on the aryloxyacetato group. The complexes show antimicrobial activity, being most efficient against Candida albicans and Bacillus subtilis.     

A New Functional Equation Analogous to CAUCHT-PEXIDER Functional Equation and its Application

The CAUCHY-PEXIDER functional equation H (x±y)=F(x) G(y) is generalized to the form H ((x^c±y^c)^1/c) = F(x) G(y), c≠0, assuming the function H(x) possesses a measurable majorant on a set of positive measure. The result is used to obtain a characterization of WEIBULL distribution. This functional equation is generalized to functions of vector variables.     

Parnassius apollo last-instar larvae development prediction by analysis of weather condition as a tool in the species’ conservation

Parnassius apollo (Lepidoptera: Papilionidae) has already disappeared or is under threat of extinction in many of its former habitats. It has been documented that weather conditions – anomalies in particular – contributed to this process. In this study, we combined developmental data obtained previously for the last-instar Apollo larvae (collected in 1996, 1997, and 2003) with corresponding meteorological data to assess the effects of ambient temperature and rainfall episodes on the duration and the completion of the instar. For comparing the temperature effect, we applied the degree-day concept. We found significant positive correlation between the number of rainy days during the instar development (x) and its duration time (y): y = 8.293 + 0.936x (± 2.813) (r = 0.662, P < 10⁻⁷). Logarithmic transformation of the growth curves of the last-instar Apollo larvae revealed that there was no difference in growth among females; however, there was slower growth of males in 2003 in comparison to 1996. Growth (y) of female Apollo larvae as a function of instar duration (x) can by described by one common equation, irrespectively of the year: y = 317.6 + 502.3 lnx (± 263.3) (r = 0.82, P < 10⁻⁴).     

Induction of Freezing Tolerance in Spinach during Cold Acclimation

Spinach (Spinacia oleracea L.) seedlings, grown in soil or on an agar medium in vitro, became cold acclimated when exposed to a constant 5°C. Plants subjected to cold acclimation, beginning 1 week postgermination, attained freezing tolerance levels similar to that achieved by seedlings that were cold acclimated beginning 3 weeks after sowing. Seedlings at 1 week of age had only cotyledonary leaves, while 3-week-old seedlings had developed true leaves. Plants grown in vitro were able to increase in freezing tolerance, but were slightly less hardy than soil-grown plants. These results suggest that spinach, a cool-season crop that begins growth in early spring when subzero temperatures are likely, can undergo cold acclimation at the earliest stages of development following germination. Axenic seedlings, grown in vitro, were used to develop a noninjurious radiolabeling technique. Leaf proteins were radiolabeled to specific activities of 10⁵ counts per minute per microgram at 25°C or 5 × 10⁴ counts per minute per microgram at 5°C over a 24 hour period. The ability to radiolabel leaf proteins of in vitro grown plants to high specific activities at low temperature, without injury or microbial contamination, will facilitate studies of cold acclimation.     

The induction of kin genes in cold-acclimating Arabidopsis thaliana. Evidence of a role for calcium

The involvement of calcium signaling during cold-induction of the kin genes of Arabidopsis thaliana (L.) Heynh. was examined. Treatments with chemicals which either chelate extracellular calcium (EGTA) or block the plasma-membrane calcium channels (La³⁺, Gd³⁺) inhibited cold acclimation as well as kin gene expression. Ruthenium red, an inhibitor of calcium release from intracellular stores partially inhibited kin gene expression and development of freezing tolerance. An inhibitor of calcium-dependent protein kinases (CDPKs) and calmodulin prevented cold acclimation as well as the cold induction of kin genes. Using restriction fragment length polymorphism-coupled domain-directed differential display, five CDPK clones were identified which showed differential regulation by cold. The amplified fragments showed homology to known plant CDPKs. The involvement of calcium and calcium-binding proteins in cold acclimation of A. thaliana is discussed. Received: 28 November 1996 / Accepted: 5 May 1997     

Three azido-, alk/phenoxido- and acetato-bridged tetranuclear nickel complexes featuring defective double-cubane

Three new tetranuclear nickel(II) complexes of general formula [Ni₄(L)₂(N₃)₂(CH₃COO)₂(CH₃O)₂]₂·xCH₃OH·yH₂O (HL = HL¹, HL² and HL³; x = 0, y = 1 for 1; x = 2, y = 0 for 2 and x = 2, y = 4 for 3) were synthesized and characterized by single crystal X-ray diffraction and magnetic measurements. Single crystal X-ray studies reveal that all three complexes exhibit similar tetranuclear face-shared defective double-cubane structure, having azido-, alk/phenoxido- and acetato-bridges. Magnetic susceptibility measurements on the complexes in the range of 300-2 K indicate ferromagnetic coupling between the metal ions. The slightly different magnetic behaviors observed are probably caused by subtle structural differences between the respective [Ni₄O₄N₂] cores induced by ligand variation.     

Adaptive character of metabolism in Eothenomys miletus in Hengduan Mountains region during cold acclimation

Environmental factors play an important role in the seasonal adaptation of body mass and thermogenesis in small, wild mammals. The purpose of the present study was to test the hypothesis that ambient temperature was a cue to trigger the seasonal adjustments in body mass, energy intake, uncoupling protein 1 (UCP1) in brown adipose tissue (BAT), and other biochemical characteristics of Eothenomys miletus during 49 days of cold exposure. Our data demonstrated that cold acclimation induced a remarkable decrease in body mass, a significant increase in energy intake and metabolic rate, and high expression of UCP1 in BAT of E. miletus. Biochemical characteristics of BAT and liver respiration were also increased following cold acclimation. These data suggest that E. miletus reduced the body mass and increased energy intake and expenditure under cold acclimation. Increased expression of UCP1 was potentially involved in the regulation of energy metabolism and thermogenic capacity following cold acclimation.     

Effects of Red and Far Red Light on the Initiation of Cold Acclimation in Cornus stolonifera Michx

Red and far red light distinctly influence the initial phytochrome-mediated phase of cold acclimation in red-osier dogwood (Cornus stolonifera). Under controlled conditions, short days and end-of-day far red light exposure after long days promote growth cessation, cold acclimation, and subsequent cold hardening of dogwood stems in response to low temperature. Nuclear magnetic resonance absorption spectra of the water in internode stem sections imply that the short day-induced phase of cold acclimation involves a change in tissue hydration, at least in part, due to a substantial reduction in bulk phase water as a result of senescence and loss of water from the pith. Seasonal responses to light and an attempt to induce early acclimation under natural conditions with end-of-day far red light are discussed.     

Preparation and separation of asymmetric tripodal iron complexes of tren and selected aldehydes

Reaction of iron salts with the tripodal ligands formed from the condensation of tris-(2-aminoethyl)amine (tren) with the following mixtures of aldehydes, 2-pyridinecarboxaldehyde (py) and 6-methyl-2-pyridinecarboxaldehyde (6-CH₃py), salicylaldehyde (sal) and 2-hydroxy-5-methylbenzaldehyde (5-CH₃sal), salicylaldehyde and pyrrole-2-carboxaldehyde (pyr), and 4-methyl-5-imidazolecarboxaldehyde (4-CH₃ImH) and salicylaldehyde yielded mixtures of all four possible iron complexes, [Fe(tren)L_xL′_y]^z (x +y = 3), where L and L′ represent different aldehydes. Preliminary indication of mixtures of products was provided by IR and UV-Vis spectroscopy. Conclusive proof of the existence of all four components (x = 0-3) in each of the reaction mixtures is provided by mass spectroscopy. Separation of the [Fetren(sal)_x(4-CH₃ImH)_y](ClO₄)_y mixture can be achieved using a Sephadex ion exchange column. Treatment of the two observed fractions with base yields greatly enriched [Fetren(sal)₂(4-CH₃Im)] and [Fetren(sal)(4-CH₃Im)₂], which were identified by mass spectroscopy.     

The effects of temperature and aging of Drosophila males on the frequencies of XXY and XO progeny

Drosophila males of the constitution y/y⁺Y were aged for 10 days at 25° and 10° and then mated daily for 13 days at 25° to virgin yw (XX) females. The total frequencies of exceptional XXY and XO offspring to which the father contributed either (a) both an X and a Y chromosome, or (b) neither of them, were highly significantly more numbers in the 7th- and 8th-day broods of the 10° than the 25° pre-aged series. In all experiments the frequency of paternal XO exceptions was greatly in excess of that of XXY exceptions. The data on brood patterns suggest that the stages most sensitive to the production of paternal exceptions by pre-aging at 10° are those of the primary spermatocytes. The same stages are also sensitive to low temperature induction of temporarily low fertility.     

Does acclimation in cavitation resistance due to mechanical perturbation support the pit area or conduit reinforcement hypotheses in Phaseolus vulgaris?

Two Phaseolus vulgaris L. cultivars were exposed to reduced water and stem mechanical perturbation treatments (flexing) to determine if acclimation to these treatments induced hydraulic changes, altered cavitation resistance and changed stem mechanical properties. Additionally, this study sought to determine if changes in cavitation resistance would support the pit area or conduit reinforcement hypotheses. Flexing reduced biomass, leaf area, xylem vessel area and hydraulic conductivity. One cultivar had greater measures of stem strength and cavitation resistance. Flexing increased cavitation resistance (P₅₀) but did not increase Young's modulus, rigidity or flexural strength on dried stems. Stem rigidity and basal diameter were correlated with leaf mass. The ratio of conduit wall thickness to span [(t/b)_h²] increased under high water and flexing treatments while rigidity decreased for one cultivar exposed to both flexing and lower water suggesting an inability to compensate for two simultaneous stresses. Although P₅₀ was not correlated with measures of mechanical strength, P₅₀ was correlated with vessel diameter, consistent with the pit area hypothesis. This study confirmed that mechanical perturbation can impact xylem structural properties and result in altered plant water flow characteristics and cavitation resistance. Long‐term hydraulic acclimation in these herbaceous annuals was constrained by similar tradeoffs that constrain hydraulic properties across species.