The biogenesis of ethylene in Penicillium digitatum

The origin of ethylene in Penicillium digitatum has been shown to be intimately associated with the Krebs cycle. 2-Ketoglutaric acid and glutamic acid are the most efficient precursors of ethylene, which is derived from carbons 3 and 4 of these substrates as a unit. However, which of these is the immediate precursor has not been established. Since 2-ketoglutaric acid is a very efficient precursor and succinic acid is an inefficient one, 2-ketoglutaric acid must be the branching point at which the pathway of ethylene biosynthesis leads away from the Krebs cycle. This conclusion is in full agreement with the following observations: Three of the four hydrogen atoms of the ethylene molecule were derived from protons of the medium; C-2 but not C-1 of acetate was incorporated into ethylene; and [2,3-¹⁴C]succinic acid but not [2,3-³H]succinic acid was incorporated.     

Inhibition of ethylene production by rhizobitoxine

Rhizobitoxine, an inhibitor of methionine biosynthesis in Salmonella typhimurium, inhibited ethylene production about 75% in light-grown sorghum seedlings and in senescent apple tissue. Ethylene production stimulated by indoleacetic acid and kinetin in sorghum was similarly inhibited. With both apple and sorghum, the inhibition could only be partially relieved by additions of methionine. A methionine analogue, α-keto-γ-methylthiobutyric acid, which has been suggested as an intermediate between methionine and ethylene, had no effect on the inhibition.     

Reducing jasmonic acid levels causes ein2 mutants to become ethylene responsive

It has previously been shown that jasmonic acid affects the ethylene signaling pathway. EIN2 is a central component of ethylene signaling that is downstream of the receptors. EIN2 has previously been shown to be required for ethylene responses. We found that reducing jasmonic acid levels, either mutationally or chemically, caused ein2 ethylene-insensitive mutants to become ethylene responsive. This effect was not seen with the ethylene-insensitive etr1-1 mutants that affect receptor function. Based upon these results, we propose a model where jasmonic acid is inhibiting ethylene signal transduction down-stream of the ethylene receptors. This may involve an EIN2-independent pathway.     

The influence of ethylene and ethylene modulators on shoot organogenesis in tomato

The influence of ethylene and ethylene modulators on the in vitro organogenesis of tomato was studied using a highly regenerating accession of the wild tomato Solanum pennellii and an F1 plant resulting from a cross between Solanum pennellii and Solanum lycopersicum cv. Anl27, which is known to have a low regeneration frequency. Four ethylene-modulating compounds, each at four levels, were used, namely: cobalt chloride (CoCl₂), which inhibits the production of ethylene; AgNO₃ (SN), which inhibits ethylene action; and Ethephon and the precursor 1-aminocyclopropane-1-carboxylic acid (ACC), which both promote ethylene synthesis. Leaf explants of each genotype were incubated on shoot induction medium supplemented with each of these compounds at 0, 10 or 15 days following bud induction. The results obtained in our assays indicate that ethylene has a significant influence on tomato organogenesis. Concentrations of ethylene lower than the optimum (according to genotype) at the beginning of the culture may decrease the percentage of explants with buds (B), produce a delay in their appearance, or indeed inhibit bud formation. This was observed in S. pennellii and the F1 explants cultured on media with SN (5.8–58.0 μM) as well as in the F1 explants cultured on medium with 21.0 μM CoCl₂. The percentage of explants with shoots (R) and the mean number of shoots per explant with shoots (PR) also diminished in media that contained SN. Shoots isolated from these explants were less developed compared to those isolated from control explants. On the other hand, ethylene supplementation may contribute to enhancing shoot development. The number of isolable shoots from S. pennellii explants doubled in media with ACC (9.8–98.0 μM). Shoots isolated from explants treated with ethylene releasing compounds showed a higher number of nodes when ACC and Ethephon were added at 10 days (in F1 explants) or at 15 days (in S. pennellii) after the beginning of culture. Thus, the importance of studying not only the concentration but also the timing of the application of regulators when developing regeneration protocols has been made manifest. An excess of ethylene supplementation may produce an inhibitory effect, as was observed when using Ethephon (17.2–69.0 μM). These results show the involvement of ethylene in tomato organogenesis and lead us to believe that ethylene supplementation may contribute to enhancing regeneration and shoot development in tomato.     

Regulation of soybean nodulation independent of ethylene signaling

Leguminous plants regulate the number of Bradyrhizobium- or Rhizobium-infected sites that develop into nitrogen-fixing root nodules. Ethylene has been implicated in the regulation of nodule formation in some species, but this role has remained in question for soybean (Glycine max). The present study used soybean mutants with decreased responsiveness to ethylene, soybean mutants with defective regulation of nodule number, and Ag⁺ inhibition of ethylene perception to examine the role of ethylene in the regulation of nodule number. Nodule numbers on ethylene-insensitive mutants and plants treated with Ag⁺ were similar to those on wild-type plants and untreated plants, respectively. Hypernodulating mutants displayed wild-type ethylene sensitivity. Suppression of nodule numbers by high nitrate was also similar between ethylene-insensitive plants, wild-type plants, and plants treated with Ag⁺. Ethylene insensitivity of the roots of etr1-1 mutants was confirmed using assays for sensitivity to 1-aminocyclopropane-1-carboxylic acid and for ethylene-stimulated root-hair formation. Additional phenotypes of etr1-1 roots were also characterized. Ethylene-dependent pathways regulate the number of nodules that form on species such as pea and Medicago truncatula, but our data indicate that ethylene is less significant in regulating the number of nodules that form on soybean.     

Current methods for detecting ethylene in plants

Background

In view of ethylene''s critical developmental and physiological roles the gaseous hormone remains an active research topic for plant biologists. Progress has been made to understand the ethylene biosynthesis pathway and the mechanisms of perception and action. Still numerous questions need to be answered and findings to be validated. Monitoring gas production will very often complete the picture of any ethylene research topic. Therefore the search for suitable ethylene measuring methods for various plant samples either in the field, greenhouses, laboratories or storage facilities is strongly motivated.

Scope

This review presents an update of the current methods for ethylene monitoring in plants. It focuses on the three most-used methods – gas chromatography detection, electrochemical sensing and optical detection – and compares them in terms of sensitivity, selectivity, time response and price. Guidelines are provided for proper selection and application of the described sensor methodologies and some specific applications are illustrated of laser-based detector for monitoring ethylene given off by Arabidopsis thaliana upon various nutritional treatments.

Conclusions

Each method has its advantages and limitations. The choice for the suitable ethylene sensor needs careful consideration and is driven by the requirements for a specific application.Key words: Ethylene, Arabidopsis thaliana, gas sampling, gas chromatography, electrochemical sensing, laser-based detector     

Bean abscission cellulase : characterization of a cDNA clone and regulation of gene expression by ethylene and auxin

The physiology and anatomy of abscission has been studied in considerable detail; however, information on the regulation of gene expression in abscission has been limited because of a lack of probes for specific genes. We have identified and sequenced a 595 nucleotide bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase cDNA clone (pBACl). The bean cellulase cDNA has extensive nucleic and amino acid sequence identity with the avocado cellulase cDNA pAV363. The 2.0 kilobase bean mRNA complementary to pBACl codes for a polypeptide of approximately 51 kilodalton (shown by hybrid-selection followed by in vitro translation). Bean cellulase antiserum is shown to immunoprecipitate a 51 kilodalton polypeptide from the in vitro translation products of abscission zone poly(A)⁺ RNA. Ethylene initiates bean leaf abscission and tissue-specific expression of cellulase mRNA. If ethylene treatment of bean explants was discontinued after 31 h and then 2,5-norbornadiene given to inhibit responses resulting from endogenously synthesized ethylene, polysomal cellulase mRNA hybridizing to pBACl decreased. Thus, ethylene is required not only to initiate abscission and cellulase gene expression but also to maintain continued accumulation of cellulase mRNA. Explants treated with auxin 4 hours prior to a 48 hour treatment with ethylene showed no substantial accumulation of RNA hybridizing to pBACl or expression of cellulase activity.     

Preparation of ethylene gas and comparison of ethylene responses induced by ethylene,ACC, and ethephon

Ethylene is a gaseous plant hormone used in many physiological studies examining its role in plant growth and development. However, ethylene gas may not be conveniently available to many laboratories for occasional use, and therefore several chemicals can be used as replacements. Here we report that the kinetics of the ethylene response induced by ethylene and two widely-used ethylene replacements are different. ACC failed to efficiently replace prolonged ethylene treatments, while the decomposition products of ethephon may cause non-specific responses and the efficiency of ethephon conversion to ethylene was relatively low. A cost-effective method to prepare ethylene gas was developed. Analyzed by gas chromatography, the chemically produced ethylene exhibited an identical chromatogram to that from the commercial source. Our synthetic ethylene gave the same dose-response curve in Arabidopsis as gaseous ethylene. Our study shows that the use of the ethylene gas is essential to experiments that are sensitive to treatment duration and dosage. When ACC and ethephon are used as replacements, caution should be taken in the experimental design. For laboratories that do not have an ethylene tank, ethylene gas can be easily prepared by a chemical approach without further purification.     

Information theory and the ethylene genetic network

The original aim of the Information Theory (IT) was to solve a purely technical problem: to increase the performance of communication systems, which are constantly affected by interferences that diminish the quality of the transmitted information. That is, the theory deals only with the problem of transmitting with the maximal precision the symbols constituting a message. In Shannon''s theory messages are characterized only by their probabilities, regardless of their value or meaning. As for its present day status, it is generally acknowledged that Information Theory has solid mathematical foundations and has fruitful strong links with Physics in both theoretical and experimental areas. However, many applications of Information Theory to Biology are limited to using it as a technical tool to analyze biopolymers, such as DNA, RNA or protein sequences. The main point of discussion about the applicability of IT to explain the information flow in biological systems is that in a classic communication channel, the symbols that conform the coded message are transmitted one by one in an independent form through a noisy communication channel, and noise can alter each of the symbols, distorting the message; in contrast, in a genetic communication channel the coded messages are not transmitted in the form of symbols but signaling cascades transmit them. Consequently, the information flow from the emitter to the effector is due to a series of coupled physicochemical processes that must ensure the accurate transmission of the message. In this review we discussed a novel proposal to overcome this difficulty, which consists of the modeling of gene expression with a stochastic approach that allows Shannon entropy (H) to be directly used to measure the amount of uncertainty that the genetic machinery has in relation to the correct decoding of a message transmitted into the nucleus by a signaling pathway. From the value of H we can define a function I that measures the amount of information content in the input message that the cell''s genetic machinery is processing during a given time interval. Furthermore, combining Information Theory with the frequency response analysis of dynamical systems we can examine the cell''s genetic response to input signals with varying frequencies, amplitude and form, in order to determine if the cell can distinguish between different regimes of information flow from the environment. In the particular case of the ethylene signaling pathway, the amount of information managed by the root cell of Arabidopsis can be correlated with the frequency of the input signal. The ethylene signaling pathway cuts off very low and very high frequencies, allowing a window of frequency response in which the nucleus reads the incoming message as a varying input. Outside of this window the nucleus reads the input message as an approximately non-varying one. This frequency response analysis is also useful to estimate the rate of information transfer during the transport of each new ERF1 molecule into the nucleus. Additionally, application of Information Theory to analysis of the flow of information in the ethylene signaling pathway provides a deeper insight in the form in which the transition between auxin and ethylene hormonal activity occurs during a circadian cycle. An ambitious goal for the future would be to use Information Theory as a theoretical foundation for a suitable model of the information flow that runs at each level and through all levels of biological organization.Key words: information theory, shannon entropy, frequency systems analysis, Arabidopsis thaliana, ethylene signaling systems, plant genetic networks, circadian cycles     

GDSL lipase 1 regulates ethylene signaling and ethylene-associated systemic immunity in Arabidopsis

Arabidopsis GDSL lipase 1 (GLIP1) has been shown to modulate systemic immunity through the regulation of ethylene signaling components. Here we demonstrate that the constitutive triple response mutant ctr1-1 requires GLIP1 for the ethylene response, gene expression, and pathogen resistance. The glip1-1 mutant was defective in induced resistance following primary inoculation of necrotrophic pathogens, whereas GLIP1-overexpressing plants showed resistance to multiple pathogens. Necrotrophic infection triggered the downregulation of EIN3 and the activation of ERF1 and SID2 in a GLIP1-dependent manner. These results suggest that GLIP1 positively and negatively regulates ethylene signaling, resulting in an ethylene-associated, necrotroph-induced immune response.     

Auxin stimulation of ethylene evolution

The stimulation of ethylene production from seedling tissue of Phascolus vulgaris, Helianthus annuus and Zea mays by growth regulators was inhibited by actinomycin D and puromycin and to a lesser extent by 2-thiouracil and p-fluorophenylalanine. It is concluded that the mechanism of action of growth regulators on the enhancement of ethylene production is the formation of enzymes involved in ethylene biogenesis.     

Dual effects of ethylene on potato dormancy and sprout growth

Dormant potato tubers (Solanum tuberosum L.) of two cultivars were treated with various concentrations of ethylene gas for various exposure periods. As has been shown by others, ethylene caused a rapid but transient increase in respiration rate, which appeared to be independent of any effects on dormancy. All concentrations tested caused accelerated sprouting, 2 microliters per liter being the most effective. Ethylene exerts a dual effect on potato tubers: it markedly shortens the duration of rest, but it inhibits elongation of the sprouts during extended treatment. Comparing these results with published work on seeds, bulbs, and corms suggests that ethylene must have a significant but as yet unexplained role in rest and dormancy. However, since the most effective ethylene treatment did not equal the response elicited by treatment with ethylene chlorhydrin, other factors must also contribute to termination of rest.     

Effect of 1-methylcyclopropene and methylenecyclopropane on ethylene binding and ethylene action on cut carnations

1-Methylcyclopropene (1-MCP), formerly designated as Sis-X, has been shown to be an effective inhibitor of ethylene responses in carnation flowers in either the light or the dark. The binding appears to be to the receptor and to be permanent. A 6 h treatment at 2.5 nl l^–1 is sufficient to protect against ethylene, and 0.5 nl l^–1 is sufficient if exposure is for 24 h. As carnation flowers age, a little higher concentration appears to be needed. Most of the natural increase in ethylene production during senescence is prevented by treatment with 1-MCP. A closely related compound, methylenecyclopropane shows ethylene activity. A tritium labelled 1-MCP (60 mCi mmol^–1) has been prepared. A higher specific activity is needed for more critical studies.     

Biosynthesis of ethylene glycol in Escherichia coli

Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d-xylose is reported. This route consists of four steps: d-xylose?→?d-xylonate?→?2-dehydro-3-deoxy-d-pentonate?→?glycoaldehyde?→?EG. Respective enzymes, d-xylose dehydrogenase, d-xylonate dehydratase, 2-dehydro-3-deoxy-d-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the d-xylose?→?d-xylulose reaction was prevented by disrupting the d-xylose isomerase gene. The most efficient construct produced 11.7 g?L^?1 of EG from 40.0 g?L^?1 of d-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde?→?glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.     

Methionine metabolism in apple tissue in relation to ethylene biosynthesis

A comparison of the rate of ethylene production by apple fruit to the methionine content of the tissue suggests that the sulfur of methionine has to be recycled during its continuous synthesis of ethylene. The metabolism of the sulfur of methionine in apple tissue in relation to ethylene biosynthesis was investigated. The results showed that in the conversion of methionine to ethylene the CH₃S-group of methionine is first incorporated as a unit into S-methylcysteine. By demethylation, S-methylcysteine is metabolized to cysteine. Cysteine then donates its sulfur to form methionine, presumably through cystathionine and homocysteine. This view is consistent with the observation that cysteine, homoserine and homocysteine were all converted to methionine, in an order of efficiency from least to greatest. For the conversion to ethylene, methionine was the most efficient precursor, followed by homocysteine and homoserine. Based on these results, a methionine-sulfur cycle in relation to ethylene biosynthesis is presented.     

Synthesis of N7-hydroxyethylguanine and O6-hydroxyethylguanine Markers for the reaction of ethylene oxide (EO) with DNA

O⁶-Hydroxyethylguanine has been synthesized by reaction of mono-sodium glycolate with 6-chloroguanine. The crystalline product has been characterized using a variety of analytical techniques and compared with a sample of the corresponding N⁷-hydroxyethyl derivative. These 2 chemicals may prove useful as standards when studying the reaction of ethylene oxide (EO) with DNA.     

Inhibition of ethylene effects in iris bulbs by 2,5-norbornadiene

Mitochondria from iris (Iris hollandica cv. Ideal) bulbs (circumference 7–8 cm) that have been treated with ethylene to evoke early flowering show an induction of alternative respiratory capacity and a rise in state 3 respiration. Mitochondria from untreated control bulbs have no alternative respiration and these bulbs produce few early flowering plants. A pretreatment with a competitive inhibitor of ethylene, 2,5-norbornadiene (NBD), inhibits ethylene effects on respiration. Effects of application of 100 ppm ethylene on respiration are not completely counteracted by 4000 ppm NBD, even after a 5-day pretreatment with NBD, but effects of application of 10 ppm ethylene are inhibited by 8000 ppm NBD after a 3-day NBD-pretreatment. The flowering percentage of 10 ppm ethylene-treated bulbs is 100%, while after a NBD treatment less than 10% of the bulbs produced flowering plants. NBD by itself did not influence the alternative pathway in mitochondria appears to be dependent on the kind of respiratory substrate used: a high capacity is found with pyruvate, a low capacity with NADH, while with succinate intermediate values are observed. The capacity of the alternative respiration is not directly correlated with state 3 respiratory activity.