Kinetic Measurements of Small Ethylene Changes in an Open System Designed for Plant Physiological Studies

Based upon the method we developed to measure ethylene in very low concentrations (as low as 0.01 ppb in the ambient atmosphere) an experimental chamber was constructed and integrated in the measuring system for plant physiological studies. Parameters influencing the accuracy of the technique are evaluated. The pressure in the plant chamber increased 0.05 atm at a flow rate of 10 1/h during ethylene trapping. At this flow rate the ethylene production per seedling is independent of the number of seedlings used. The ethylene measured per seedling is directly proportional to the length of trapping time. Under standard conditions of chamber configuration and volume very small changes in ethylene content can be detected accurately in a very short time range. Our experimental arrangement allows kinetic studies of ethylene evolution by biological objects in a qualitative and quantitative manner. All methods used heretofore are more complicated and less accurate compared to the measuring system presented here. Its versatility is demonstrated for both in vitro and in vivo studies of ethylene production by bean seedlings. The application fields of the apparatus are discussed.     

Rapid Kinetic Analysis of Ethylene Growth Responses in Seedlings: New Insights into Ethylene Signal Transduction

Ethylene is a phytohormone that influences diverse processes in plants. Ethylene causes various changes in etiolated seedlings that differ between species and include reduced growth of shoots and roots, increased diameter of shoots, agravitropic growth, initiation of root hairs, and increased curvature of the apical hook. The inhibition of growth in etiolated seedlings has become widely used to screen for and identify mutants. This approach has led to an increased understanding of ethylene signaling. Most studies use end-point analysis after several days of exposure to assess the effects of ethylene. Recently, the use of time-lapse imaging has re-emerged as an experimental method to study the rapid kinetics of ethylene responses. This review outlines the historical use of ethylene growth kinetic studies and summarizes the recent use of this approach coupled with molecular biology to provide new insights into ethylene signaling.     

钙在植物乙烯生成及信号传递中的生理作用

Ｃａ＾２＋对植物乙烯生成的调节与作用位点有关,胞外Ｃａ＾２＋在维持质膜功能的同时,抑制乙烯生成,延缓衰老;过量Ｃａ＾２＋进入胞质,胞内Ｃａ＾２＋促进乙烯生成和衰老。内源ＣａＭ与乙烯生成关系密切,介入了乙烯代谢和外源激素对乙烯的调控。此外,Ｃａ＾２＋是乙烯信号传递所必需的。     

Ethylene oxide: evaluation of genotoxicity data and an exploratory assessment of genetic risk

A risk estimate of the heritable effects of ethylene oxide exposure, using the parallelogram approach, as suggested by Frits Sobels, is described. The approach is based on available data on the ethylene oxide-induced responses for the same genetic endpoint in somatic cells of both laboratory animals and humans, and for germ cell mutations in the same laboratory animal. Human germ cell effects are estimated. The available data sets for this approach were evaluated. We consider this as complementary to the genetic risk assessment carried out by U.S. EPA scientists, in which the risk from heritable (reciprocal) translocations induced by ethylene oxide was estimated. In the present study we restricted our assessment to dominant mutations. The sensitivity factor relating mouse to man was based on ethylene oxide-induced HPRT mutant frequencies in lymphocytes in vivo. From this comparison, it could be concluded that occupational exposure for 1 year to 1 ppm ethylene oxide would lead to a risk of a dominantly inherited disease in the offspring of 4 × 10⁻⁴ above the background level. The uncertainty interval of this figure is quite large (0.6–28) × 10⁻⁴. The values are compatible with the existing estimates of the corresponding risk from exposure to low LET radiation when the genotoxic potency ratio of ethylene oxide and radiation is considered. This risk estimation approach has allowed us to identify additional data that are required for a more complete risk estimation of the heritable effects of ethylene oxide, or indeed any mutagenic chemical.     

Ethylene as an endogenous inhibitor of root regeneration in tomato leaf discs cultured in vitro

We examined ethylene effects on root regeneration in tomato leaf discs cultured in vitro. Applied ethylene or Ethephon did not stimulate rooting in the leaf discs. In the presence of indoleacetic acid. 5 × 10^-6M, these substances significantly inhibited root formation. Ethylene production (nl C₂H₄· (24 h)^-1. flask^-1) was positively correlated with increased IAA concentrations at various times during the culture period and, as a consequence, with the rooting response after 168 h. However, separate testing of equimolar concentrations of seven different auxins and auxin-like compounds showed no positive correlation between the rate of ethylene production and subsequent rooting response. Aeration of gas-tight flasks containing leaf discs and absorption of ethylene evolved from the discs by mercuric perchlorate in gas-tight flasks or pre-treatment of leaf discs with AgNO₃ significantly enhanced IAA induced root regeneration. Thus, these studies indicate that ethylene is not a rooting hormone per se. Furthermore, ethylene (whether applied externally or synthesized by the tissue) does not appear to account for the ability of auxin to stimulate rooting.     

Kinetics of intracellular carbon allocation in a marine diatom

To test models of intracellular carbon flow we measured the labelling kinetics (from ¹⁴CO₂) of major classes of cell polymers (carbohydrate, protein, lipid) and of dissolved organic carbon produced by the marine diatom Thalassiosira pseudonana Hustedt, grown at rates of 0.2 to 2.0·day^?1 under nitrogen or light limitation. Compartmental analysis indicated that tracer carbon quickly entered respiratory and excretory streams, accumulating in the cells at the rate of net production after only 25–50% of cell generation (doubling) time. Respiration rates were low (≤ 0.1 · day^?1) and suggested that illuminated cells in steady-state growth made only minor use of oxidative respiration to support cell synthesis. The tracer was quick to enter all polymers; compartmental analysis indicated that polymer labelling rates were close to the rates of mass synthesis after several hours of incubation with ¹⁴C. Polymer labelling also showed a reallocation of photosynthate from protein to carbohydrate within a few hours of perturbation (shift-down) of nutrient supply in a N-limited chemostat. In steady-state growth, the protein: carbohydrate ratio increased directly with N-limited growth rate but attained its maximum under extreme light-limitation. Carbon flow into the metabolic processes of respiration, excretion and polymer synthesis appeared to be mediated by a small and rapidly cycled pool of substrates under all steady-state growth conditions.     

Kinetics of protein agglomeration. A nephelometric method for the determination of total protein in biological samples

The kinetics of agglomeration of proteins precipitating in a viscous solution was measured by light scattering. The resulting transitory maximum was linearly proportional to the mass of protein over three orders of magnitude. The change in scattering intensity is described as a change in scattering symmetry due to a continuous in particle size.This method is both fast (minutes) and sensitive (30 ng protein) and is independent of the chemical composition of the different protein species and is barely influenced by size and shape of the proteins.By solubilising the protein in an alkaline dedecyl sulfate solution this method can be applied to all types of biological samples (e.g. tissue homogenates proteins) and also to all types of biological preparations containing detergents as well as urea, sucrose, salts and lipids.     

Circadian Ethylene Synthesis in <Emphasis Type="Italic">Sorghum bicolor</Emphasis>: Expression and Control of the System at the Whole Plant Level

Ethylene production by sorghum is rhythmic and the amplitude of the rhythm is increased both by dim, far-red enriched light and in mutant plants deficient in phytochrome B. The mechanisms involved in controlling ethylene production were examined in detail by measuring the rate of ethylene production among organs and tissues, examining the organ-specific levels of ACC (1-aminocyclopropane-1-carboxylic acid, the ethylene precursor) and investigating the contribution of the roots to shoot ethylene production. The results demonstrate that the expanding leaves were the major source of ethylene under dim, far-red enriched light and in the phytochrome B mutant. Enhanced ethylene production by the expanding leaf appeared to be the result of targeted delivery of ACC to this tissue. Root ACC levels were much higher than those in the shoot but roots converted much less of this endogenous ACC to ethylene. Applying ACC to the roots had only a marginal effect on their ethylene production, but greatly increased that of the shoots. Decapitated shoots continued to produce ethylene in a rhythmic pattern but the amplitude decreased with time compared to intact plants. The results collectively suggest that some, but not all, of the shoot ethylene rhythm depends on the transport of ACC from the roots to the shoots.     

超级高产小麦旗叶荧光动力学研究

探明超级小麦品种的旗叶光合作用与荧光动力学特性,为超级小麦品种选育利用提供理论依据。以超级小麦临麦4号为试验材料,应用CI-301PS型便携式光合作用测定系统和FMS-2便携式荧光测定仪(英国Hansatech公司)在田间试验中测定旗叶光合作用与荧光动力学参数。结果表明,与普通高产对照品种皖麦52和烟农19相比,超级小麦临麦4号的光合作用参数光合速率、光饱和点和CO2饱和点、羧化效率高,光补偿点和CO2补偿点低;光合机构系统工作参数PSII实际的光化学效率(ΦPSII)、光化学猝灭系数(qP)、PSII反应中心的激发能捕获效率(Fv/Fm)、PSⅡ潜在活性Fv/Fo和电子传递速率(ETR)值高,非光化学猝灭系数(NPQ)值低。这表明超级小麦临麦4号的光合机构系统工作能力强和工作效率高,保证旗叶光合作用的高效运行,为子粒灌浆提供充足的能量和碳水化合物。     

Kinetics of Encapsulated Yeast Alcohol Dehydrogenase Dispersed in an Organic Solvent

Microcapsules dispersed in organic solvents provide a suitable environment for conducting enzyme reactions involving cofactors and hydrophobic substrates. Encapsulated YADH is active and stable in cyclohexane provided the pH is adjusted appropriately. Mass transfer does not influence batch reaction rates. Conversion in a fluidized-bed reactor containing encapsulated YADH/NAD⁺ and employing cyclohexane as the continuous phase depends strongly on residence time and inlet cinnamyl alcohol concentration. However, interpretation of these results is complicated by enzyme inactivation by the product, cinnamaldehyde, and interference from residual encapsulating agents.     

Kinetics of Encapsulated Yeast Alcohol Dehydrogenase Dispersed in an Organic Solvent

Microcapsules dispersed in organic solvents provide a suitable environment for conducting enzyme reactions involving cofactors and hydrophobic substrates. Encapsulated YADH is active and stable in cyclohexane provided the pH is adjusted appropriately. Mass transfer does not influence batch reaction rates. Conversion in a fluidized-bed reactor containing encapsulated YADH/NAD⁺ and employing cyclohexane as the continuous phase depends strongly on residence time and inlet cinnamyl alcohol concentration. However, interpretation of these results is complicated by enzyme inactivation by the product, cinnamaldehyde, and interference from residual encapsulating agents.     

Ethylene induces zygote formation through an enhanced expression of zyg1 in Dictyostelium mucoroides

We have previously demonstrated that a potent plant hormone, ethylene induces sexual development including zygote formation in Dictyostelium cells, and that a novel gene (zyg1) is also involved in zygote formation. Based on these findings, the present work was mainly designed to reveal (1) the precise relationship between the ethylene amount and zygote formation, and (2) the relation of in situ ethylene synthesis to zyg1 expression, using transformants that over- or under-produce ACC-oxidase (Dd-aco) involved in ethylene biosynthesis. ACO(OE) cells overexpressing Dd-aco gene overproduced ethylene and exhibited the augmented zygote formation. In contrast, ACO-RNAi cells, in which the expression of Dd-aco was suppressed by the RNAi method, showed a reduced level of ethylene production, thus resulting in inhibition of zygote formation. Importantly, the expression of zyg1 was affected by the amount of ethylene produced: Zyg1 expression was augmented in ACO(OE) cells, but was significantly suppressed in ACO-RNAi cells. In another experiment, we found that 1-methylcyclopropene (1-MCP), which is known to inhibit the function of ethylene by binding specifically to ethylene receptors, greatly suppresses zygote formation. These results indicate that ethylene is capable of inducing zygote formation through the expression of zyg1.     

Ethylene and growth control in the fringed waterlily (Nymphoides peltata): Stimulation of cell division and interaction with buoyant tension in petioles

The effect of ethylene on petiole growth of the Fringed Waterlily (Nymphoides peltata (S.G. Gmelin) O. Kuntze) changes during leaf ontogeny. During early development (before expansion of laminae), ethylene causes an increase in both cell number and cell size; later in development, promotion of rapid cell expansion is the dominant effect. The early effects may contribute to the accommodation of new leaves to water columns of different depth. The later effects on cell expansion only are shown to contribute to the rapid accommodation of floating leaves when changes in water level submerge the laminae. This kind of accommodation results from an interaction between accumulated ethylene, which increases wall extensibility, and the tension in petioles due to natural buoyancy which, it is suggested, supplements the driving force for cell expansion. Cell age (position) within a petiole and age of the whole petiole influence the growth response to ethylene alone and the amount of extra growth produced by applying tension when ethylene is present. In young petioles, apical cells are highly sensitive to ethylene and tension causes little further growth; older cells in both immature and mature petioles show little response to ethylene unless the petiole is under tension. Young (but not mature) petioles respond slowly to applied tension even in the absence of ethylene. It is concluded that as cells age the driving force for expansion limits increasingly their capacity to respond to the wall-loosening effects of ethylene. Dual sensitivity to ethylene and buoyant tension facilitates rapid accommodation responses but sensitivity of young petioles to tension alone may exclude Nymphoides from habitats where current velocity is appreciable.     

Responses of the Steroid Circadian System to Alcohol in Humans: Importance of the Time and Duration of Intake

Reports provide conflicting data about the effects of alcohol consumption on the hormonal system. Any study of these effects must control for a number of variables, including sex, alcohol status (alcoholic addiction vs. non-addiction), medical status (malnutrition, liver disease), and conditions of alcohol exposure, including an acute or continuous pattern of intake. The latter appears to be an especially critical factor in interpreting these effects. The authors therefore conducted a trial with a circadian design in which alcohol was administered repeatedly and regularly over a 26 h period for a total dose of 256 g. Because this protocol involves continuous alcohol administration, it is similar to administration among alcoholics and thus sheds new light on alcohol's effect on hormone secretion. Using healthy volunteers rather than alcoholics, however, prevents any confounding due to liver disorders and nutritional deficiencies, and thus makes it possible to focus on the direct role of alcohol in hormonal modifications. In these conditions, the continuous administration of alcohol did not affect cortisol secretion, but serum testosterone levels were significantly higher at all time points during the alcohol session than at the corresponding time points during the control session. These data are not consistent with previously reported findings for the relation between alcohol and both cortisol and testosterone, because in the current experiment the action of ethanol on steroid secretion should involve the circadian clock more than the hormonal system itself.     

Macroinvertebrate Assemblages of an Andean High‐Altitude Tropical Stream: The Importance of Season and Flow

We studied the Piburja stream, a high‐altitude tropical stream in Ecuador. Our main goals were to determine whether the macroinvertebrate community composition and abundance differed between seasons, reaches and velocity patches. Likewise, we aimed to examine the importance of the hydrological regime in determining these differences. Flow was significantly higher in the wet season, but the stability of flow was higher in the dry season. There was a strong increase in macroinvertebrate community metrics (richness, density and diversity) in the dry season. Seasons and velocity patches better explained the community composition. Reaches did not show differences at the community level, but some taxa showed significant differences among reaches. Our findings differed from those published in previous studies that have suggested that mountain tropical streams are non‐seasonal. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Prediction of the Quantity and Purity of an Antibody Capture Process in Real Time

Regulatory recommendations for quality by design instead of quality by testing raise increasing interest in new sensor technologies. An online monitoring system for downstream processes is developed, which is based on an array of online detectors. Besides standard detectors (UV, pH, and conductivity), our chromatographic workstation is equipped with a fluorescence and a mid‐infrared spectrometer, a light scattering, and a refractive index detector. The combination of these sensors enables the prediction of specific protein concentration and various purity attributes, such as high molecular weight impurities, DNA and host cell protein content during the elution phase of a chromatographic antibody capture process. Prediction models solely based on online signals are set up providing real‐time predictions. No mechanistic models or information about the chromatographic runs is used. These predictions allow online pooling decisions replacing time‐ and labor‐intensive laboratory measurements. Different process variations, such as changes in the column load or elution buffer, are introduced to test the predictive power of the models. Extrapolation of the models worked well when the column load is changed, whereas model adjustment is necessary when the elution conditions are changed considerably.     

Anatomy of a pulmonary immune response: Kinetics in different leukocyte pools

In pulmonary immune reactions the cells which can be obtained by bronchoalveolar lavage (BAL) are only one part of the picture. In this study the kinetics of an experimental pulmonary immune response were investigated simultaneously in different lung compartments in the same rat. On days 0, 1, 2, 3, 4, 5 and 11 after intratracheal challenge with sheep red blood cells, leukocytes were taken from the bronchoalveolar, the interstitial and the marginal lung vascular pool as well as from the peripheral blood. Total numbers of granulocytes, NK cells, B and T cells, CD4⁺ and CD8⁺ cells were determined. Histology and in vivo labeling of proliferating cells was performed. On day 1 after challenge an increase of granulocytes in the BAL was found. In the BAL the total number of T lymphocytes increased on day 1 and day 2 and the CD4/CD8 ratio increased from day 1 to day 5, indicating an influx of CD4⁺ T cells. Changes in the lung interstitium showed a similar tendency, but were not found in the marginal pool or blood. Histologically cellular infiltrates were seen around the pulmonary small vessels. Little local proliferation occurred in the different lung compartments, indicating mainly immigration of cells. Further studies will focus on the expression of adhesion molecules during an immune response, to learn more about the mechanisms responsible for the increase of lymphocytes.     

Dry-Heat Destruction of Bacillus subtilis Spores on Surfaces: Effect of Humidity in an Open System

Bacillus subtilis var. niger spores were tested for dry-heat resistance on stainless-steel strips hung in an oven. Heat resistance was dependent on the relative humidity before and during treatment, which in turn affected the water content of the spores. Higher humidities increased the heat resistance of the spores. D-values ranged from 16.1 min for spores conditioned at <2% relative humidity (RH) and treated at 0.34% RH to 37.6 min for spores conditioned at 89% RH and treated at 1.1% RH. The y-intercept of the regression line ranged from 6.94 x 10(4) for spores conditioned and treated at the low humidities to 2.00 x 10(5) for spores conditioned at 89% RH and treated at 0.34% RH. For a constant value of N(0), the y-intercept appears to be lowered by low-humidity conditions. The statistic log y(0)/log N(0) is used to measure the downward displacement of the regression line. Values obtained in this experiment range from 0.90 for spores conditioned at <2% RH and treated at 0.34% RH to 1.04 for spores conditioned at <2% RH and treated at 1.1% RH. A combination of linear regression and analysis of variance methods was used for data analysis. The former estimates D-values and y-intercepts, whereas the latter is sensitive to differences between treatments.