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

<p>Abstract</p> <p>Background</p> <p>The 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.</p> <p>Results</p> <p>This 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 <it>in vitro </it>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.</p> <p>Conclusions</p> <p>This 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.</p

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

Background: Solanum 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. Results: All 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. Conclusions: The Ethylene biosynthesis pathway and E8 show a tissue specific and developmental differentiation throughout tomato fruit development and ripening

Ethylene biosynthesis of 'Jonagold' apple during storage and shelf life: A modeling approach

With a yearly production of 300,000 ton, apple is an important Belgian product with "Jonagold" being the main cultivar. Apple is a climacteric fruit and is susceptible to an ethylene regulated ripening process which causes the fruit to become softer, less crispy and develop a greasy skin.To control the ripening process, apples are stored under controlled atmosphere (CA) conditions after harvest, often in combination with a treatment with ethylene inhibitors like 1-MCP (SmartfreshTM). Under these conditions the O2- and CO2- partial pressure are controlled in such a way that the respiration and the associated quality changes are reduced to a minimum, without introducing undesired physiological disorders.The optimal combination of storage conditions depends on the species, cultivar and ripeness stage and is to be determined experimentally. This requires a substantial amount of time and storage infrastructure. If this experimental approach could be supported by a model based simulation and decision making system, the sector would greatly benefit from it. For this purpose a mathematical model is needed that contains sub-models describing gas transport, respiration, the ethylene metabolism and fruit quality. The objective of this thesis was the development of a kinetic model that describes the ethylene production in "Jonagold" apple under varying temperature and atmospheric conditions. Further, this modelwas chosen to be based on the known physiology of the ethylene biosynthesis and on experimental data on all relevant enzymes and metabolites.In a first step the necessary analytical measuring protocols were optimized. The developed protocols are a combination of existing protocols which were standardized and scaled down where possible and new protocols, which replaced old and time consuming protocols.Subsequently a large storage experiment was performed during two consecutive harvest years. The objective of these experiments was to determine the effect of both harvest date and treatment with 1-MCP on the ethylene biosynthesis and quality of apple. The ethylene biosynthesisof untreated apples during CA storage was found to depend on the harvest date, but this dependency decreased during shelf life. The effect of the treatment with 1-MCP was stronger than the effect of harvest date and lasted for the entire storage and shelf life period. Based on the quantitative analysis a clear picture was obtained of all components of the ethylene biosynthesis in "Jonagold" apple during CA storage and shelf life.The obtained data served as the basis for the estimation of the 31 model parameters of a newly developed kinetic ethylene model. The developed model was able to adequately describe the ethylene production of "Jonagold" apple during shelf life as well as CA storage. Additionally, the model allowed for an improved understanding of the regulation of the ethylene biosynthesis. The metabolite SAM proved not to be rate limiting for the biosynthesis of ethylene. The regulation of the enzymes ACS and ACO differed greatly. ACS activity was almost completely suppressed during storage while the ACO activity increased steadily. Based on the model results it was postulated that the difference in activity was a consequence of differences in expression levels of genes coding for both enzymes during both storage and shelf life. The results revealed a need for information concerning fast changes of the ethylene biosynthesis inresponse to drastic changes in the ambient conditions. In an independent experiment, online continuous ethylene measurements were combined with destructive sampling for the analysis of enzymesand metabolites at discrete moments in time. For these samples, not only enzyme activity but also protein levels and gene expression was determined. Both a fast, 12-24 hours lasting response and a slower,longer lasting response of the ethylene biosynthesis was observed upon changing temperature and atmosphere conditions. The fast response was presumably caused by the adaption of the fruit to the changing conditions while the slower response was due to long term changes at the transcriptional and translational level. The changes in ACS activity were mainly associated with changes in the expression of ACS1 which was regulating for the cold induced peak in ethylene production. During shelf life the peak in ACO1 expression caused the increase in ACO activity necessary for the exponential increase of ethylene production during the climacteric ripening. Although ACO was never rate limiting we saw some indications of post-translational control at the end of shelf life.The developed model can be used for the prediction of the ethylene production in both storage and shelf life. This, in turn, can be used as an input for other models, e.g., that describe the loss of firmness intime. The next step will be the integration of the ethylene model with existing respiration models to form a detailed gas-exchange model that can be used for the prediction of the storage potential of individual apple batches.Dankwoord i Abstract iv Samenvatting vii Abbreviations and Symbols x Contents xiii 1 Introduction 1 1.1 Ethylene, the driving force of fruit ripening . . . . . . . . 1 1.2 Firmness as a measure for apple quality . . . . . . . . . . 4 1.3 Apple harvest and storage . . . . . . . . . . . . . . . . . . 5 1.4 Problem statement . . . . . . . . . . . . . . . . . . . . . . 10 1.5 Objective of the thesis . . . . . . . . . . . . . . . . . . . . 11 1.6 Outline of the thesis . . . . . . . . . . . . . . . . . . . . . 12 2 Literature Study 13 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2.2 Ethylene biosynthesis . . . . . . . . . . . . . . . . . . . . . 14 2.3 Ethylene perception and signal transduction . . . . . . . . 20 2.4 Ethylene inhibition . . . . . . . . . . . . . . . . . . . . . . 25 2.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3 Measuring protocols 31 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 31 3.2 Ethylene measurement (Protocol 1) . . . . . . . . . . . . . 32 3.3 Quantification of ACC and MACC (Protocol 2) . . . . . . 34 3.4 In vitro activity of ACS (Protocol 3) . . . . . . . . . . . . 41 3.5 In vitro activity of ACO (Protocol 4) . . . . . . . . . . . . 45 3.6 Quantification of SAM . . . . . . . . . . . . . . . . . . . . 49 3.7 List of chemicals used in the protocols . . . . . . . . . . . 50 3.8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . 52 4 Postharvest ripening of "Jonagold" apple 53 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 53 4.2 Literature review . . . . . . . . . . . . . . . . . . . . . . . 54 4.3 Materials and methods . . . . . . . . . . . . . . . . . . . . 56 4.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 4.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 4.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . 77 5 Ethylene biosynthesis model 79 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 79 5.2 Literature review . . . . . . . . . . . . . . . . . . . . . . . 80 5.3 Materials and methods . . . . . . . . . . . . . . . . . . . . 82 5.4 Model development . . . . . . . . . . . . . . . . . . . . . . 85 5.5 Model calibration . . . . . . . . . . . . . . . . . . . . . . . 94 5.6 Model sensitivity to fclim,0 . . . . . . . . . . . . . . . . . . 107 5.7 Model validation . . . . . . . . . . . . . . . . . . . . . . . 109 5.8 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 5.9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . 117 6 Dynamic changes of the ethylene biosynthesis 119 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 119 6.2 Literature review . . . . . . . . . . . . . . . . . . . . . . . 120 6.3 Materials and methods . . . . . . . . . . . . . . . . . . . . 121 6.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 6.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 6.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . 148 7 General Conclusions 151 7.1 General conclusions . . . . . . . . . . . . . . . . . . . . . . 151 7.2 Remarks for future research . . . . . . . . . . . . . . . . . 155 References 157 List of Publications 181nrpages: 210status: publishe

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.published_online: 2014-01-21status: publishe

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

The use of capillary electrophoresis to identify ethylene precursors (ACC, MACC & SAM) in fruit tissue

status: publishe

S-adenosyl-L-methionine (SAM) usage during climacteric ripening of tomato in relation to ethylene and polyamine biosynthesis and transmethylation capacity

S-adenosyl-L-methionine (SAM) is the major methyl donor in cells and it is also used for the biosynthesis of polyamines and the plant hormone ethylene. During climacteric ripening of tomato (Solanum lycopersicum 'Bonaparte'), ethylene production rises considerably which makes it an ideal object to study SAM involvement. We examined in ripening fruit how a 1-MCP treatment affects SAM usage by the three major SAM-associated pathways. The 1-MCP treatment inhibited autocatalytic ethylene production but did not affect SAM levels. We also observed that 1-(malonylamino)cyclopropane-1-carboxylic acid formation during ripening is ethylene dependent. SAM decarboxylase expression was also found to be upregulated by ethylene. Nonetheless polyamine content was higher in 1-MCP-treated fruit. This leads to the conclusion that the ethylene and polyamine pathway can operate simultaneously. We also observed a higher methylation capacity in 1-MCP-treated fruit. During fruit ripening substantial methylation reactions occur which are gradually inhibited by the methylation product S-adenosyl-L-homocysteine (SAH). SAH accumulation is caused by a drop in adenosine kinase expression, which is not observed in 1-MCP-treated fruit. We can conclude that tomato fruit possesses the capability to simultaneously consume SAM during ripening to ensure a high rate of ethylene and polyamine production and transmethylation reactions. SAM usage during ripening requires a complex cellular regulation mechanism in order to control SAM levels.status: publishe

S-Adenosyl-l-methionine usage during climacteric ripening of tomato in relation to ethylene and polyamine biosynthesis and transmethylation capacity

S-adenosyl-l-methionine (SAM) is the major methyl donor in cells and it is also used for the biosynthesis of polyamines and the plant hormone ethylene. During climacteric ripening of tomato (Solanum lycopersicum Bonaparte'), ethylene production rises considerably which makes it an ideal object to study SAM involvement. We examined in ripening fruit how a 1-MCP treatment affects SAM usage by the three major SAM-associated pathways. The 1-MCP treatment inhibited autocatalytic ethylene production but did not affect SAM levels. We also observed that 1-(malonylamino)cyclopropane-1-carboxylic acid formation during ripening is ethylene dependent. SAM decarboxylase expression was also found to be upregulated by ethylene. Nonetheless polyamine content was higher in 1-MCP-treated fruit. This leads to the conclusion that the ethylene and polyamine pathway can operate simultaneously. We also observed a higher methylation capacity in 1-MCP-treated fruit. During fruit ripening substantial methylation reactions occur which are gradually inhibited by the methylation product S-adenosyl-l-homocysteine (SAH). SAH accumulation is caused by a drop in adenosine kinase expression, which is not observed in 1-MCP-treated fruit. We can conclude that tomato fruit possesses the capability to simultaneously consume SAM during ripening to ensure a high rate of ethylene and polyamine production and transmethylation reactions. SAM usage during ripening requires a complex cellular regulation mechanism in order to control SAM levels

Dynamic changes of the ethylene biosynthesis in ‘Jonagold’ apple

In this study, the short-term and dynamic changes of the ethylene biosynthesis of Jonagold apple during and after application of controlled atmosphere (CA) storage conditions were quantified using a systems biology approach. Rapid responses to imposed temperature and atmospheric conditions were captured by continuous online photoacoustic ethylene measurements. Discrete destructive sampling was done to understand observed changes of ethylene biosynthesis at the transcriptional, translational and metabolic level. Application of the ethylene inhibitor 1-methylcyclopropene (1-MCP) allowed for the discrimination between ethylene-mediated changes and ethylene-independent changes related to the imposed conditions. Online ethylene measurements showed fast and slower responses during and after application of CA conditions. The changes in 1-aminocyclopropane-1-carboxylate synthase (ACS) activity were most correlated with changes in ACS1 expression and regulated the cold-induced increase in ethylene production during the early chilling phase. Transcription of ACS3 was found ethylene independent and was triggered upon warming of CA-stored apples. Increased expression of ACO1 during shelf life led to a strong increase in 1-aminocyclopropane-1-carboxylate oxidase (ACO) activity, required for the exponential production of ethylene during system 2. Expression of ACO2 and ACO3 was upregulated in 1-MCP-treated fruit showing a negative correlation with ethylene production. ACO activity never became rate limiting.status: publishe