Bram Van de Poel

Conservation of ethylene as a plant hormone over 450 million years of evolution.

Land plants evolved more than 450 million years ago from a lineage of freshwater charophyte green algae(1). The extent to which plant signalling systems existed before the evolutionary transition to land is unknown. Although charophytes occupy a key phylogenetic position for elucidating the origins of such signalling systems(2-4), there is a paucity of sequence data for these organisms(5,6). Here we carry out de novo transcriptomics of five representative charophyte species, and find putative homologues for the biosynthesis, transport, perception and signalling of major plant hormones. Focusing on the plant hormone ethylene, we provide evidence that the filamentous charophyte Spirogyra pratensis possesses an ethylene hormone system homologous to that in plants. Spirogyra produces ethylene and exhibits a cell elongation response to ethylene. Spirogyra ethylene-signalling homologues partially rescue mutants of the angiosperm Arabidopsis thaliana and respond post-translationally to ethylene when expressed in plant cells, indicative of unambiguously homologous ethylene-signalling pathways in Spirogyra and Arabidopsis. These findings imply that the common aquatic ancestor possessed this pathway prior to the colonization of land and that cell elongation was possibly an ancestral ethylene response. This highlights the importance of charophytes for investigating the origins of fundamental plant processes.

1-aminocyclopropane-1-carboxylic acid (ACC) in plants: more than just the precursor of ethylene!

Ethylene is a simple two carbon atom molecule with profound effects on plants. There are quite a few review papers covering all aspects of ethylene biology in plants, including its biosynthesis, signaling and physiology. This is merely a logical consequence of the fascinating and pleiotropic nature of this gaseous plant hormone. Its biochemical precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) is also a fairly simple molecule, but perhaps its role in plant biology is seriously underestimated. This triangularly shaped amino acid has many more features than just being the precursor of the lead-role player ethylene. For example, ACC can be conjugated to three different derivatives, but their biological role remains vague. ACC can also be metabolized by bacteria using ACC-deaminase, favoring plant growth and lowering stress susceptibility. ACC is also subjected to a sophisticated transport mechanism to ensure local and long-distance ethylene responses. Last but not least, there are now a few exciting studies where ACC has been reported to function as a signal itself, independently from ethylene. This review puts ACC in the spotlight, not to give it the lead-role, but to create a picture of the stunning co-production of the hormone and its precursor.

Protocol: An updated integrated methodology for analysis of metabolites and enzyme activities of ethylene biosynthesis

BackgroundThe foundations for ethylene research were laid many years ago by researchers such as Lizada, Yang and Hoffman. Nowadays, most of the methods developed by them are still being used. Technological developments since then have led to small but significant improvements, contributing to a more efficient workflow. Despite this, many of these improvements have never been properly documented.ResultsThis article provides an updated, integrated set of protocols suitable for the assembly of a complete picture of ethylene biosynthesis, including the measurement of ethylene itself. The original protocols for the metabolites 1-aminocyclopropane-1-carboxylic acid and 1-(malonylamino)cyclopropane-1-carboxylic acid have been updated and downscaled, while protocols to determine in vitro activities of the key enzymes 1-aminocyclopropane-1-carboxylate synthase and 1-aminocyclopropane-1-carboxylate oxidase have been optimised for efficiency, repeatability and accuracy. All the protocols described were optimised for apple fruit, but have been proven to be suitable for the analysis of tomato fruit as well.ConclusionsThis work collates an integrated set of detailed protocols for the measurement of components of the ethylene biosynthetic pathway, starting from well-established methods. These protocols have been optimised for smaller sample volumes, increased efficiency, repeatability and accuracy. The detailed protocol allows other scientists to rapidly implement these methods in their own laboratories in a consistent and efficient way.

Targeted Systems Biology Profiling of Tomato Fruit Reveals Coordination of the Yang Cycle and a Distinct Regulation of Ethylene Biosynthesis during Postclimacteric Ripening

The concept of system 1 and system 2 ethylene biosynthesis during climacteric fruit ripening was initially described four decades ago. Although much is known about fruit development and climacteric ripening, little information is available about how ethylene biosynthesis is regulated during the postclimacteric phase. A targeted systems biology approach revealed a novel regulatory mechanism of ethylene biosynthesis of tomato (Solanum lycopersicum) when fruit have reached their maximal ethylene production level and which is characterized by a decline in ethylene biosynthesis. Ethylene production is shut down at the level of 1-aminocyclopropane-1-carboxylic acid oxidase. At the same time, 1-aminocyclopropane-1-carboxylic acid synthase activity increases. Analysis of the Yang cycle showed that the Yang cycle genes are regulated in a coordinated way and are highly expressed during postclimacteric ripening. Postclimacteric red tomatoes on the plant showed only a moderate regulation of 1-aminocyclopropane-1-carboxylic acid synthase and Yang cycle genes compared with the regulation in detached fruit. Treatment of red fruit with 1-methylcyclopropane and ethephon revealed that the shut-down mechanism in ethylene biosynthesis is developmentally programmed and only moderately ethylene sensitive. We propose that the termination of autocatalytic ethylene biosynthesis of system 2 in ripe fruit delays senescence and preserves the fruit until seed dispersal.

Ethylene and Hormonal Cross Talk in Vegetative Growth and Development

Recent insights into the role of ethylene in regulating plant growth and development highlight interactions with other hormones. Ethylene is a gaseous plant hormone that most likely became a functional hormone during the evolution of charophyte green algae, prior to land colonization. From this ancient origin, ethylene evolved into an important growth regulator that is essential for myriad plant developmental processes. In vegetative growth, ethylene appears to have a dual role, stimulating and inhibiting growth, depending on the species, tissue, and cell type, developmental stage, hormonal status, and environmental conditions. Moreover, ethylene signaling and response are part of an intricate network in cross talk with internal and external cues. Besides being a crucial factor in the growth control of roots and shoots, ethylene can promote flowering, fruit ripening and abscission, as well as leaf and petal senescence and abscission and, hence, plays a role in virtually every phase of plant life. Last but not least, together with jasmonates, salicylate, and abscisic acid, ethylene is important in steering stress responses.

FERONIA receptor kinase interacts with S-adenosylmethionine synthetase and suppresses S-adenosylmethionine production and ethylene biosynthesis in Arabidopsis

Environmental inputs such as stress can modulate plant cell metabolism, but the detailed mechanism remains unclear. We report here that FERONIA (FER), a plasma membrane receptor-like kinase, may negatively regulate the S-adenosylmethionine (SAM) synthesis by interacting with two S-adenosylmethionine synthases (SAM1 and SAM2). SAM participates in ethylene, nicotianamine and polyamine biosynthetic pathways and provides the methyl group for protein and DNA methylation reactions. The Arabidopsis fer mutants contained a higher level of SAM and ethylene in plant tissues and displayed a dwarf phenotype. Such phenotype in the fer mutants was mimicked by over-expressing the S-adenosylmethionine synthetase in transgenic plants, whereas sam1/2 double mutant showed an opposite phenotype. We propose that FER receptor kinase, in response to environmental stress and plant hormones such as auxin and BR, interacts with SAM synthases and down-regulates ethylene biosynthesis.

Tissue specific analysis reveals a differential organization and regulation of both ethylene biosynthesis and E8 during climacteric ripening of tomato

BackgroundSolanum lycopersicum or tomato is extensively studied with respect to the ethylene metabolism during climacteric ripening, focusing almost exclusively on fruit pericarp. In this work the ethylene biosynthesis pathway was examined in all major tomato fruit tissues: pericarp, septa, columella, placenta, locular gel and seeds. The tissue specific ethylene production rate was measured throughout fruit development, climacteric ripening and postharvest storage. All ethylene intermediate metabolites (1-aminocyclopropane-1-carboxylic acid (ACC), malonyl-ACC (MACC) and S-adenosyl-L-methionine (SAM)) and enzyme activities (ACC-oxidase (ACO) and ACC-synthase (ACS)) were assessed.ResultsAll tissues showed a similar climacteric pattern in ethylene productions, but with a different amplitude. Profound differences were found between tissue types at the metabolic and enzymatic level. The pericarp tissue produced the highest amount of ethylene, but showed only a low ACC content and limited ACS activity, while the locular gel accumulated a lot of ACC, MACC and SAM and showed only limited ACO and ACS activity. Central tissues (septa, columella and placenta) showed a strong accumulation of ACC and MACC. These differences indicate that the ethylene biosynthesis pathway is organized and regulated in a tissue specific way. The possible role of inter- and intra-tissue transport is discussed to explain these discrepancies. Furthermore, the antagonistic relation between ACO and E8, an ethylene biosynthesis inhibiting protein, was shown to be tissue specific and developmentally regulated. In addition, ethylene inhibition by E8 is not achieved by a direct interaction between ACO and E8, as previously suggested in literature.ConclusionsThe Ethylene biosynthesis pathway and E8 show a tissue specific and developmental differentiation throughout tomato fruit development and ripening.

Determination of S-adenosyl-l-methionine in fruits by capillary electrophoresis.

INTRODUCTION S-adenosyl-l-methionine (SAM) plays an important role in many biochemical reactions in plants. It is mainly used as a methyl donor for methylation reactions, but it also participates in, for example, the biosynthesis of polyamines and the plant hormone ethylene. OBJECTIVE To develop a fast capillary electrophoresis technique to separate SAM in fruits and fruit juices without any pre-purification steps. METHODOLOGY Four different extraction solutions and two extraction times were tested, of which 5% trichloroacetic acid (TCA) for 10 min was found most suited. A glycine : phosphate buffer (200 : 50 mm, pH 2.5) was found optimal to analyse SAM in TCA extracts. Analyses were preformed on different climacteric and non-climacteric fruits and fruit juices. The calibration curve was created in degraded tomato extract. The CE-method was compared with a more conventional HPLC method described in literature. RESULTS The CE technique made it possible to completely separate the S,S- and R,S-diastereoisomeric forms of SAM. The CE method proved to be very fast (20 min total running time instead of 42 min) and more sensitive (limit of detection of 0.5 µm instead of 1 µm) compared with the conventional HPLC method. CONCLUSION Fast measurements of SAM in fruits and juices are favoured by capillary electrophoresis in a 200 : 50 mm glycine : phosphate (pH 2.5) buffer system.

A transcriptomics-based kinetic model for ethylene biosynthesis in tomato (Solanum lycopersicum) fruit: development, validation and exploration of novel regulatory mechanisms

The gaseous plant hormone ethylene is involved in many physiological processes including climacteric fruit ripening, in which it is a key determinant of fruit quality. A detailed model that describes ethylene biochemistry dynamics is missing. Often, kinetic modeling is used to describe metabolic networks or signaling cascades, mostly ignoring the link with transcriptomic data. We have constructed an elegant kinetic model that describes the transfer of genetic information into abundance and metabolic activity of proteins for the entire ethylene biosynthesis pathway during fruit development and ripening of tomato (Solanum lycopersicum). Our model was calibrated against a vast amount of transcriptomic, proteomic and metabolic data and showed good descriptive qualities. Subsequently it was validated successfully against several ripening mutants previously described in the literature. The model was used as a predictive tool to evaluate novel and existing hypotheses regarding the regulation of ethylene biosynthesis. This bottom-up kinetic network model was used to indicate that a side-branch of the ethylene pathway, the formation of the dead-end product 1-(malonylamino)-1-aminocyclopropane-1-carboxylic acid (MACC), might have a strong effect on eventual ethylene production. Furthermore, our in silico analyses indicated potential (post-) translational regulation of the ethylene-forming enzyme ACC oxidase.

Transcriptome Profiling of the Green Alga Spirogyra pratensis (Charophyta) Suggests an Ancestral Role for Ethylene in Cell Wall Metabolism, Photosynthesis, and Abiotic Stress Responses.

The plant hormone ethylene regulates predominantly cell wall metabolism, photosynthesis, and abiotic stress responses in the charophyte green alga Spirogyra pratensis. It is well known that ethylene regulates a diverse set of developmental and stress-related processes in angiosperms, yet its roles in early-diverging embryophytes and algae are poorly understood. Recently, it was shown that ethylene functions as a hormone in the charophyte green alga Spirogyra pratensis. Since land plants evolved from charophytes, this implies conservation of ethylene as a hormone in green plants for at least 450 million years. However, the physiological role of ethylene in charophyte algae has remained unknown. To gain insight into ethylene responses in Spirogyra, we used mRNA sequencing to measure changes in gene expression over time in Spirogyra filaments in response to an ethylene treatment. Our analyses show that at the transcriptional level, ethylene predominantly regulates three processes in Spirogyra: (1) modification of the cell wall matrix by expansins and xyloglucan endotransglucosylases/hydrolases, (2) down-regulation of chlorophyll biosynthesis and photosynthesis, and (3) activation of abiotic stress responses. We confirmed that the photosynthetic capacity and chlorophyll content were reduced by an ethylene treatment and that several abiotic stress conditions could stimulate cell elongation in an ethylene-dependent manner. We also found that the Spirogyra transcriptome harbors only 10 ethylene-responsive transcription factor (ERF) homologs, several of which are regulated by ethylene. These results provide an initial understanding of the hormonal responses induced by ethylene in Spirogyra and help to reconstruct the role of ethylene in ancestral charophytes prior to the origin of land plants.