Ethylene signal transduction during climacteric tomato fruit ripening

Tomato (Solanum lycopersicum L.) is a crop of high economic and nutritional value produced worldwide. Besides having a high number of vitamins and minerals, tomato fruit is rich in other bioactive components including carotenoids and phenolic compounds. In addition, it is an ideal model system to study fleshy fruit development and ripening thanks to its relative short life cycle, its small genome size and the availability of a large selection of germplasms and mutants. The ripening of tomato, and of climacteric fruit in general, is regulated by the plant hormone ethylene, which also regulates numerous other aspects of plant growth and development including responses to biotic and abiotic stress. Postharvest control of ethylene is of great importance to assure proper ripening conditions and to control fruit quality. Thus, a good understanding of ethylene perception by the fruit is essential to eventually improve postharvest practices.

The objective of this thesis is to gain insight into the dynamic changes in the ethylene signal transduction during climacteric ripening of tomato, in order to, in the long term, improve the quality and storability of tomato after harvest.

Ethylene is perceived by the ethylene receptors (ETRs) which are the first steps in the complex pathway of the ethylene signal transduction. Receptors, together with the CTR proteins inhibit the action of EIN2, a key regulator of the signalling pathway which activates ethylene-related transcription factors in the nucleus. A gene expression analysis was carried out using RT-qPCR of SlETRs and SlCTRs during tomato fruit ripening on-vine as compared to ripening off-vine either or not treated with the ethylene inhibitor 1-methylcyclopropene (1-MCP). Off-vine postharvest ripening showed a different regulation than on-vine ripening, while treatment with 1-MCP inhibited the expression of some genes more than others. The results suggested that some genes were more ethylene-responsive than others but that cooperatively might control the timing of ripening.

To fully understand the underlying regulation and physiological responses, there was a need to complement these transcriptomic results with the quantification of the proteins, which are the real effectors of the reactions. However, there was not yet a well-established way to identify and quantify these proteins, with only some of the receptors being quantified through western blots. Our approach started as an MS discovery analysis in a highly fractionated tomato peptide sample which allowed the identification of 8,588 proteins, one of the largest dataset described for Solanum lycopersicum L. so far. The complete protein dataset was annotated through GO and KEGG databases, mapping the proteins by their cellular location and biological process. Among all the identified proteins, four tomato ethylene receptors, three SlCTRs and SlEIN2 could also be identified.

This information was taken as the starting point to create a targeted MS proteomic assay based on the parallel reaction monitoring (PRM) mode. As a result, all seven SlETRs, three out of the four SlCTRs and SlEIN2 could be identified and quantified. Their protein and mRNA levels were quantified in four ripening stages of tomato fruit. Their behaviour revealed that some of the receptors and SlCTRs may be responsible for initiating the ethylene signalling and with that ripening, while others for controlling the progress of ripening. The levels of SlEIN2, both mRNA and proteins, decreased during ripening, which might be related to its proteolysis and translocation to the nucleus upon ethylene binding to the receptors.

Finally, we wanted to further investigate the regulation of the ethylene signalling during ripening at different temperatures. For that a large storage experiment was developed in which mature green tomatoes were stored postharvest at three different temperatures and sampled every certain intervals. The analysis performed on the samples were colour, firmness, ethylene and respiration production rate, gene expression and protein quantification of the ethylene signalling elements ETRs, CTRs and EIN2, ACS and ACO enzyme activities and ACC and MACC accumulation. The results helped us to better understand the regulation of the ethylene signalling through an omics approach and to link it with the ethylene biosynthesis pathway.

As a conclusion, we have developed a feasible and reproducible assay to identify and quantify ethylene signalling proteins in tomato pericarp, which can be used by other researchers. On the other hand, we have characterised the receptors, SlCTRs and SlEIN2 using a multiomics approach which casts more light on the regulation of the ethylene signal transduction mechanism during climacteric ripening of tomato.