Morphological analysis of seed shape in Arabidopsis thaliana reveals altered polarity in mutants of the ethylene signaling pathway

The shape of Arabidopsis thaliana dry seed is described here as a prolate spheroid. The accuracy of this approximation is discussed. Considering its limitations, it allows a geometric approximation to the analysis of changes occurring in seed shape during imbibition prior to seed germination as well as the differences in shape between genotypes and their changes during imbibition. The triple mutant ein2-1, ers1-2, etr1-7 presents notable alterations in seed shape. In addition, seeds of this and other mutants in the ethylene signaling pathway (ctr1-1, eto1-1, etr1-1, ein2-1) show different response to imbibition than the wild type. Imbibed seeds of the wild type increase their asymmetry compared with the dry seeds. This is detected by the relative changes in the curvature values in both poles. Thus, during imbibition of the wild-type seeds, the reduction in curvature values observed in the basal pole gives them an ovoid shape. In contrast, in the seeds of the ethylene mutants, reduction in curvature values occurs in both basal and apical poles, and its shape remains as a prolate spheroid. Our data indicate that the ethylene signaling pathway is involved, in general, in the complex regulation of seed shape and, in particular, in the establishment of polarity in seeds, controlling curvature values in the seed poles.     

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.     

Ethylene enhances water transport in hypoxic aspen

Water transport was examined in solution culture grown seedlings of aspen (Populus tremuloides) after short-term exposures of roots to exogenous ethylene. Ethylene significantly increased stomatal conductance, root hydraulic conductivity (L(p)), and root oxygen uptake in hypoxic seedlings. Aerated roots that were exposed to ethylene also showed enhanced L(p). An ethylene action inhibitor, silver thiosulphate, significantly reversed the enhancement of L(p) by ethylene. A short-term exposure of excised roots to ethylene significantly enhanced the root water flow (Q(v)), measured by pressurizing the roots at 0.3 MPa. The Q(v) values in ethylene-treated roots declined significantly when 50 microM HgCl(2) was added to the root medium and this decline was reversed by the addition of 20 mM 2-mercaptoethanol. The results suggest that the response of Q(v) to ethylene involves mercury-sensitive water channels and that root-absorbed ethylene enhanced water permeation through roots, resulting in an increase in root water transport and stomatal opening in hypoxic seedlings.     

Limitations of the use of plant material grown in Petri dishes for physiological experiments

Roots of plants growing "aeroponically" (AP) on moistened filter paper in Petri dishes for a few days are fairly often used for physiological experiments (e.g. measurement of root growth), for ion or herbicide uptake tests, before the establishment of hydroponic or aseptic cultures although their hormonal status is markedly different from that of the hydroponic (HP) control. On the 4th day of germination the ethylene production of cucumber (Cucumis sativus L. cv. Budai csemege) roots growing in AP under controlled conditions increased considerably and exhibited a maximum curve, HP roots evolved ethylene much more constantly. The morphological changes in AP roots (e.g. inhibited elongation and swelling of primary roots, and increased formation of root hairs), resembling those caused by exogenously applied ethylene, can be prevented with 10(-5) M Ag+, an inhibitor of ethylene action. In roots of one-week-old AP seedlings, the amount of an acidic inhibitor, which as judged from the Rf values is likely to be abscisic acid (ABA), is about twice as high as in HP seedlings. An elevated ethylene or ABA level of AP roots may result in a reduced elongation of the primary roots. Counteraction of this inhibition by Ag+ suggests that the effect of ethylene is the primary event in the reduction of root length. When using plant material grown in Petri dishes the possibility of similar changes in hormonal status of the roots must be taken into consideration.     

Comparison of mRNA levels of three ethylene receptors in senescing flowers of carnation (Dianthus caryophyllus L.)

Three ethylene receptor genes, DC-ERS1, DC-ERS2 and DC-ETR1, were previously identified in carnation (Dianthus caryophyllus L.). Here, the presence of mRNAs for respective genes in flower tissues and their changes during flower senescence are investigated by Northern blot analysis. DC-ERS2 and DC-ETR1 mRNAs were present in considerable amounts in petals, ovaries and styles of the flower at the full-opening stage. In the petals the level of DC-ERS2 mRNA showed a decreasing trend toward the late stage of flower senescence, whereas it increased slightly in ovaries and was unchanged in styles throughout the senescence period. However, DC-ETR1 mRNA showed no or little changes in any of the tissues during senescence. Exogenously applied ethylene did not affect the levels of DC-ERS2 and DC-ETR1 mRNAs in petals. Ethylene production in the flowers was blocked by treatment with 1,1-dimethyl-4-(phenylsulphonyl)semicarbazide (DPSS), but the mRNA levels for DC-ERS2 and DC-ETR1 decreased in the petals. DC-ERS1 mRNA was not detected in any cases. These results indicate that DC-ERS2 and DC-ETR1 are ethylene receptor genes responsible for ethylene perception and that their expression is regulated in a tissue-specific manner and independently of ethylene in carnation flowers during senescence.     

Continuous non-destructive determination of nitrogenase activity in microbiol cultures.

The dinitrogen fixing enzyme nitrogenase (nitrogen:(acceptor) oxidoreductase)(EC 1.7.99.2) is monitored by its ability to reduce acetylene to ethylene. Low, non-inhibitory concentrations of acetylene (approximately 10(-7)mol/litre) are mixed with the gas flow aerating microbiol cultures, and acetylene and ethylene in the effluent gas are determined by gas chromatography. The procedure is safe, simple and carried out in situ without disturbing the growing culture. Transient changes in nitrogenase activity are easily detected. The technique may be automated.     

Ethylene responses in Arabidopsis seedlings include the reduction of curvature values in the root cap

Recently, curvature was described as a new trait useful in the analysis of root apex shape. Treating the root profile as a geometric curve revealed that root apex curvature values are lower in ethylene-insensitive mutants (Cervantes E, Tocino A. Geometric analysis of Arabidopsis root apex reveals a new aspect of the ethylene signal transduction pathway in development. J Plant Physiol 2005;162:1038-45). This fact suggests that curvature is regulated by ethylene. In this work, we have determined the curvature values in embryonic roots of wild-type Columbia as well as in ethylene signal-transduction mutants, and found smaller values in embryos of the mutants. We also report on the evolution of root curvature during early development after seed germination. The line Lt16b that expresses GFP in the cell wall has allowed us to investigate the evolution of curvature values in three successive cell layers of seedling roots by confocal microscopy. Treatment of seedlings with norbornadiene resulted in lower curvature values. Our results show details illustrating the effect of ethylene in root curvature.     

Role of Ethylene Metabolism in Amaranthus retroflexus

¹⁴C-Ethylene was metabolized by etiolated pigweed seedlings (Amaranthus retroflexus L.) in the manner similar to that observed in other plants. The hormone was oxidized to ¹⁴CO₂ and incorporated into ¹⁴C-tissue components. Selected cyclic olefins with differing abilities to block ethylene action were used to determine if ethylene metabolism in pigweed is necessary for ethylene action. 2,5-Norbornadiene and 1,3-cyclohexadiene were effective inhibitors of ethylene action at 800 and 6400 microliters per liter, respectively, in the gas phase, while 1,4-cyclohexadiene and cyclohexene were not. However, all four cyclic olefins inhibited the incorporation and conversion of ¹⁴C-ethylene to ¹⁴CO₂ by 95% with I₅₀ values below 100 microliters per liter. The results indicate that total ethylene metabolism does not directly correlate with changes in ethylene action. Additionally, the fact that inhibition of ethylene metabolism by the cyclic olefins did not result in a corresponding increase in ethylene evolution indicates that ethylene metabolism does not serve to significantly reduce endogenous ethylene levels.     

The influence of mechanical impedance on the growth of maize roots

Summary Maize roots were grown between 1 mm glass beads on which a pressure of 40 kPa was applied. The roots were supplied with a constant flow of aerated nutrient solution. Compared with roots grown in a nutrient solution, the impeded crown roots showed a reduction in length of about 75%, whereas the diameter was about 50% increased.These changes in root morphology have been attributed to changes in cell wall structure of the cortex cells, which also occur as a result of the influence of ethylene.It is suggested that ethylene acts as an intermediate factor in the effect of mechanical impedance on root growth.     

Ethylene production by carob (Ceratonia siliqua) callus cultures on varying media

Carob callus from hypocotyl segments produced ethylene in different amounts which were related to the composition of the medium and age of the callus. Both light and darkness stimulated high levels of ethylene production. No correlation was found between growth rate and ethylene production under dark conditions. In the light, a significant correlation was found, indicating that ethylene production and growth rate follow one another. Culture medium was the most important factor in controlling the growth rate and ethylene production. The highest values of ethylene production were obtained on media showing highest callus growth rate. These studies seem to indicate that most of the ethylene produced is a by-product of metabolic changes during carob callus development, though, under certain conditions, the initial evolution could regulate growth.     

Ethylene-induced gene expression in carnation petals : relationship to autocatalytic ethylene production and senescence

Exposure of carnation (Dianthus caryophyllus L.) flowers to ethylene evokes the developmental program of petal senescence. The temporal relationship of several aspects of this developmental program following treatment with ethylene was investigated. Exposure of mature, presenescent flowers to 7.5 microliters per liter ethylene for at least 6 hours induced petal in-rolling and premature senescence. Autocatalytic ethylene production was induced in petals following treatment with ethylene for 12 or more hours. A number of changes in mRNA populations were noted in response to ethylene, as determined by in vitro translation of petal polyadenylated RNA. At least 6 mRNAs accumulated following ethylene exposure. The molecular weights of their in vitro translation products were 81, 58, 42, 38, 35, and 25 kilodaltons. Significant increases in abundance of most mRNAs were observed 3 hours following ethylene exposure. Ethylene exposure resulted in decreased abundance of another group of mRNAs. Treatment of flowers with competitive inhibitors of ethylene action largely prevented the induction of these ethylene responses in petals. An increase in flower age was accompanied by an increase in the capacity for ethylene to induce petal in-rolling, autocatalytic ethylene production, and changes in mRNA populations suggesting that these responses are regulated by both sensitivity to ethylene and ethylene concentration. These results indicate that changes in petal physiology resulting from exposure to ethylene may be the result of rapid changes in gene expression.     

Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. III. The role of ethylene

While ethylene is suspected to be one of the many factors that play a role in rooting, some studies have found that ethylene can promote rooting, while others show it to inhibit this process or to have no effect. Using seedlings of sunflower ( Helianthus annuus L. cv. Dahlgren 131) we carried out observations on the rates of ethylene production and the levels of the ethylene precursor, 1-aminocyclopropane-l-car-boxylic acid (ACC), and ACC conjugate, l-(malonylamino)cyclopropane-l-car-boxylic acid (MACC), during the process of root initiation. The changes in these substances in the base of the hypocotyls (the portion that produces roots) were compared to the changes that occurred in the top of the hypocotyls (non-rooting portion). We also supplied a number of presumptive inhibitors of ethylene biosynthesis and inhibitors of ethylene action for short periods during the early and critical stages of root formation. Their effects on ethylene action, synthesis and rooting were examined. We conclude that the wound-induced increase in ethylene, seen within 3 h of production of the cuttings, is a key stimulatory factor in the formation of root primordia. When this increase in ethylene is localized in the lower portion of the hypocotyl, there is a promotion of rooting. On the other hand, higher concentrations in the top of the hypocotyls (as compared to the bottom) may inhibit rooting.     

Aerenchyma formation in the wetland plant Juncus effusus is independent of ethylene

Flooded plant roots commonly form aerenchyma, which allows gas diffusion between shoots and roots. The programmed cell death involved in this induced aerenchyma formation is controlled by the plant hormone ethylene, as has been shown for maize (Zea mays). However, the role of ethylene is uncertain in wetland species that form constitutive aerenchyma (also under nonflooded conditions). The aim of this study is to shed light on the involvement of ethylene in constitutive aerenchyma formation in Juncus effusus. Plants of J. effusus and maize were treated with ethylene and inhibitors of ethylene action to determine the consequences for aerenchyma formation. Neither treatment with high ethylene concentrations nor with ethylene inhibitors resulted in changes in root aerenchyma in J. effusus. By contrast, ethylene increased aerenchyma development in maize unless ethylene action inhibitors were applied simultaneously. Similarly, root elongation was insensitive to ethylene treatment in J. effusus, but was affected negatively in maize. The data show that aerenchyma in J. effusus is highly constitutive and, in contrast to the inducible aerenchyma in maize, is not obviously controlled by ethylene.     

The Effect of the Date of Rindite Treatment on the Concentration of Potato Virus Y in Tubers

The main purpose of this work was to study the host-parasite relationship between attacking fungi and the infected climacteric fruit. The study dealt mainly with physiological changes such as respiration and ethylene evolution pattern occurring in fruit inoculated with different fungi after picking. These physiological changes in infected fruit were found to be basically similar to those of uninfected fruit, but they were substantially accelerated. A direct correlation was obtained between the rate of fungal development in fruit (incubation period and rot development) and the extent of acceleration of respiration and ethylene evolution; in general, a faster development of the fungus was related to greater acceleration of ethylene and respiration rate of the infected fruit. Fungal infection was found to have an effect on fruits similar to that of exogenic ethylene treatment on sound fruit.     

Hormones and root-shoot relationships in flooded plants — an analysis of methods and results

Two aspects of root to shoot communication in flooded plants are discussed (i) the formation of porous aerenchyma that enhances the passage of oxygen, and other gases, from shoots to roots and (ii) the movement of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) from roots to shoots in the transpiration stream, and the effect of this on ethylene production and epinastic curvature in the shoots. For aerenchyma studies a highly sensitive photoacoustic laser detector for ethylene was used to avoid interference associated with other methods of ethylene measurement that require tissue excision. ACC concentrations in xylem sap were measured by physico-chemical means to ensure correct identification and account for processing losses. Solute concentrations, e.g., abscisic acid (ABA), in xylem sap are shown to be distorted by temporary contamination caused by the method used to collect sap. Concentrations of solutes in xylem sap (e.g., ACC) are also altered by changes in sap flow brought about by conventional methods of sap collection or by experimental treatments such as flooding the soil. Ways of for overcoming these problems are described together with a summary of preliminary results.     

The coupling of ethylene biosynthesis to a transmembrane,electrogenic proton flux

It is proposed that the reactions which lead to the generation of ethylene from 1-aminocyclopropane—1-carboxylic acid are arranged asymmetrically in the plasma membrane of plant cells so that ethylene biosynthesis is coupled to an inwardly directed, electrogenic flow of protons. According to this model a membrane potential (outside positive is required for ethylene biosynthesis. This proposed requirement is indicated by previous observations of a marked sensitivity of ethylene biosynthesis to the protonophore 2,4-dinitrophenol, and by its unusually strict dependence on membrane integrity.     

Possible coupling of chemical to structural dynamics in subtilisin BPN' catalyzed hydrolysis

The viscosity dependence of enzymatic catalysis was examined in subtilisin BPN' catalyzed hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-thiobenzyl ester. The viscosity of the reaction medium was varied by added glycerol, ethylene glycol, sucrose, glucose, fructose, poly(ethylene glycol) and Ficoll-400. Responses of the Michaelis-Menten parameters associated with hydrolysis were calculated from data obtained by spectrophotometric techniques. The reactions with these two substrates have catalytic rates well below the diffusion-controlled limit and thus enable us to study the viscosity effects on catalytic steps of non-transport nature. It was found that the Km values for both amide and ester reactions remained relatively independent of cosolvents. On the other hand, while the kcat values for amide were insensitive to cosolvents, those for ester were substantially attenuated except in the case of poly(ethylene glycol). The observed rate attenuations cannot be explained by changes in proton activity, water activity, dielectric constant of the reaction medium or shifts of any kinetically important pKa. Instead, the results can be adequately described by microviscosity effects on the unimolecular deacylation step with a coupling constant of 0.65 +/- 0.11. In addition, the different viscosity dependence in the acylation vs deacylation step can be rationalized in terms of fluctuation-dependent chemical dynamics of proton transfers in the context of the Bogris-Hynes model.     

Computational model for simulation of vascular adaptation following vascular access surgery in haemodialysis patients

An important number of surgical procedures for creation of vascular access (VA) in haemodialysis patients still results in non-adequate increase in blood flow (non-maturation). The rise in blood flow in arteriovenous shunts depends on vascular remodelling. Computational tools to predict the outcome of VA surgery would be important in this clinical context. The aim of our investigation was then to develop a 0D/1D computational model of arm vasculature able to simulate vessel wall remodelling and related changes in blood flow. We assumed that blood vessel remodelling is driven by peak wall shear stress. The model was calibrated with previously reported values of radial artery diameter and blood flow after end-to-end distal fistula creation. Good agreement was obtained between predicted changes in VA flow and in arterial diameter after surgery and corresponding measured values. The use of this computational model may allow accurate vascular surgery planning and ameliorate VA surgery outcomes.     

Rhythmicity in cotton seedlings

Ethylene production by detached cotyledons of cotton (Gossypium hirsutum L.) seedlings grown under cycles of 12 h darkness and 12 h light has been shown to be rhythmic, with a minimum and maximum 4 and 16 h, respectively after the start of the cycle (Rikin, Chalutz and Anderson, 1984, Plant Physiol. 75, 493–495). Treatment with silver ions stimulated the rhythmic ethylene production in both regular and inverted cycles (i.e. dark period changed to light period, and vice versa). The rate of the conversion of [3,4-¹⁴C]methionine into ethylene also followed the stimulation of rhythmic ethylene evolution by silver ions in both regular and inverted cycles, while treatment with aminoethoxyvinylglycine (AVG) decreased this stimulation. Conversion of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) into ethylene was not affected by silver ions, but was dependent upon the immediate light conditions, regardless of the time in the light-dark cycle, light decreasing and darkness increasing this process. It is concluded that silver ions stimulate the normal rhythmic ethylene production, and this stimulation is regulated at a step prior to the conversion of ACC into ethylene. The rhythmicity in other processes (cotyledon movement, phenylalanine ammonia-lyase activity, resistance to the herbicide 3-isopropyl-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide [bentazon]) was not affected by a decrease in the rhythmic changes in ethylene production by AVG or interference in ethylene action by silver ions. Thus, these rhythmic changes were not regulated by the rhythmic changes in ethylene production.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethyoxyvinylglycine - PAL phenylalanine ammonia-lyase