Periodic Reporting for period 1 - CallMechanics (The Impact of Callose Metabolism on the Mechanical Properties of Cell Wall during Tomato Ripening .)
Periodo di rendicontazione: 2020-08-01 al 2022-07-31
Tomato, Solanum lycopersicum L. is one of the most important cultivated crop plants worldwide (Klee and Giovannoni, 2011). Tomato fruit has high quality nutritional values and is an important source of nutrients ( Raúl et al. 2016). Around 177 million tonnes of tomatoes are produced per year in the world . However, around 25-42% of the yield is lost during postharvest (FAOSTAT, 2014). Rapid softening and pathogen attack are the main causes reducing the shelf-life of the fruit. Therefore, delaying these processes is one of the major targets in fruit breeding programmes
Tomato is a well-studied model of fleshy fruit. Previous research on this model revealed that softening involves changes in CW integrity, in cell turgor, fruit-water status, hydrostatic pressure, the accumulation and distribution of osmotically active solutes among other physiological process (Goulao & Oliveira, 2008) . Research on this area focuses on introducing changes in the expression of specific cell wall modifying enzymes (CWME) that target major cell wall components (such as pectins and cellulose) but there is a lack of knowledge on how minor components (such as callose) can affect fruit traits such as firmness, cell turgor or accumulation or distribution of osmotically active solutes (callose can alter symplastic communication).
In this direction, we proposed to undertake a biotechnological and genetic approach to modify callose levels in the latest stages of fruit development. The modification would result in a modification of the callose degradation in the last developmental phases of the ripening, that could modify cell wall mechanical properties, improving the texture, firmness and, in general, the shelf-life of cultivated tomatoes. In this context, our specific objectives were:
1). To generate tomato plants with modified callose levels at the latest developmental phases of fruit ontogeny.
2). To characterize changes in cell wall composition related to differences in callose levels.
3). To determine the physic-mechanical properties of fruit cell walls with different callose levels.
4). To determine the impact of callose on the development and the softening process of tomato fruits
With regards to the results of the project , the current outcome is a novel bioinformatic tool and knowledge to use in the selection of genes involved in fruit ripening. As a long-time outcome, we also expect to obtain plants producing fruits that maintain their firmness for a longer period of time, thus with reduced susceptibility to mechanical damage and pathogen attack during the postharvest period. This achievement will have a positive impact on the European Union economy by optimizing processes such as the frequency of harvesting, the handling and the transport procedures. It will also impact on human health and food security by reducing losses and the spreading of pathogens that thrive in mature soft fruits.
Tomato is a well-studied model of fleshy fruit. Previous research on this model revealed that softening involves changes in CW integrity, in cell turgor, fruit-water status, hydrostatic pressure, the accumulation and distribution of osmotically active solutes among other physiological process (Goulao & Oliveira, 2008) . Research on this area focuses on introducing changes in the expression of specific cell wall modifying enzymes (CWME) that target major cell wall components (such as pectins and cellulose) but there is a lack of knowledge on how minor components (such as callose) can affect fruit traits such as firmness, cell turgor or accumulation or distribution of osmotically active solutes (callose can alter symplastic communication).
In this direction, we proposed to undertake a biotechnological and genetic approach to modify callose levels in the latest stages of fruit development. The modification would result in a modification of the callose degradation in the last developmental phases of the ripening, that could modify cell wall mechanical properties, improving the texture, firmness and, in general, the shelf-life of cultivated tomatoes. In this context, our specific objectives were:
1). To generate tomato plants with modified callose levels at the latest developmental phases of fruit ontogeny.
2). To characterize changes in cell wall composition related to differences in callose levels.
3). To determine the physic-mechanical properties of fruit cell walls with different callose levels.
4). To determine the impact of callose on the development and the softening process of tomato fruits
With regards to the results of the project , the current outcome is a novel bioinformatic tool and knowledge to use in the selection of genes involved in fruit ripening. As a long-time outcome, we also expect to obtain plants producing fruits that maintain their firmness for a longer period of time, thus with reduced susceptibility to mechanical damage and pathogen attack during the postharvest period. This achievement will have a positive impact on the European Union economy by optimizing processes such as the frequency of harvesting, the handling and the transport procedures. It will also impact on human health and food security by reducing losses and the spreading of pathogens that thrive in mature soft fruits.
Three major milestones were achieved during this period (6 months): Bioinformatic analysis, Gene expression analysis using qRT-PCR and generation of vectors. The first part based on bioinformatics analysis has been published in Proceeding. Perry et al 2020. A summary of the findings is explained below.
The aim of the bioinformatic analysis was to identify specific cell wall β-1,3-glucanases expressed during tomato fruit ripening. 50 β -1,3- glucanases (BG) were identified in-silico using bioinformatic tools. In the publication we show phylogenetic analysis of β -1,3- glucanases (BG) of Arabidopsis thaliana and Solanum Lycopersicum. The phylogenetic analysis revealed that tomato genes are distributed in three clusters (α, β and γ) with evolutionary relations previously characterized in the model Arabidopsis thaliana. In Arabidopsis, cluster α comprises mainly the proteins previously identified to localize at cell wall microdomains named plasmodesmata (PDBG), while pathogenesis and stress responsive apoplastic enzymes are usually grouped in cluster β and γ. This suggests that tomato enzymes in clusters α may function at plasmodesmata, thus controlling cell-to-cell signalling, while genes included in cluster γ could be related to pathogenesis. Transcriptome analysis of tomato showed two types of regulation for BGs expressed in tomato fruit: enzymes in cluster α decreased their expression during the ripening, while enzymes in clusters β and γ increased their expression in mature fruits. The results suggest differential regulation for enzymes in different phylogenetic clusters suggesting evolutionary divergences in function (Perry et al 2020). In order to corroborate the transcriptomic results, expression analysis using qRT-PCR were performed. qRT-PCR expression analysis of genes belonging to the cluster α: Solyc12g0558840, Solyc02080660 and Solyc01g005830 showed a similar trend as the expression analysis from microarray data . The expression of these genes is decreasing during the fruit development. Contrary, the expression of the genes Solyc11g068440 and Solyc04g016470, belonging to the clade γ, are increasing during the development. Based on these results, functional analysis of these genes was designed. Vectors with the gene Solyc12g0558840 and Solyc11g068440 under ubiquitin constitutive promotor were performed. Experiment is still in process.
Dissemination & exploitation was a fundamental part of the fellowship in order to contribute socially and economically to Europe. However, most of the conferences and workshops expected during the last year were cancel or postponed. Alternatively, the beneficiary was attended to electronic conferences as 1st International Electronic Conference on Plant Science. And this summer will attend to the 7th International Conference on Plant Cell Wall Biology https://www.pcwb2021.com/(si apre in una nuova finestra) and Results from Marie Curie fellow will be presented.
A virtual format “ Be Curious 2020” was attended and participated in order to divulge science to the general public . outreach activities(si apre in una nuova finestra).
Currently there is a publication of the project: Louisa Perry, Yoselin Benitez-Alfonso and Candelas Paniagua “An In-Silico Approach to Identify Ripening Related beta-1,3- Glucanases and Their Role in Tomato Fruit” in the open access journal Proceedings.
The aim of the bioinformatic analysis was to identify specific cell wall β-1,3-glucanases expressed during tomato fruit ripening. 50 β -1,3- glucanases (BG) were identified in-silico using bioinformatic tools. In the publication we show phylogenetic analysis of β -1,3- glucanases (BG) of Arabidopsis thaliana and Solanum Lycopersicum. The phylogenetic analysis revealed that tomato genes are distributed in three clusters (α, β and γ) with evolutionary relations previously characterized in the model Arabidopsis thaliana. In Arabidopsis, cluster α comprises mainly the proteins previously identified to localize at cell wall microdomains named plasmodesmata (PDBG), while pathogenesis and stress responsive apoplastic enzymes are usually grouped in cluster β and γ. This suggests that tomato enzymes in clusters α may function at plasmodesmata, thus controlling cell-to-cell signalling, while genes included in cluster γ could be related to pathogenesis. Transcriptome analysis of tomato showed two types of regulation for BGs expressed in tomato fruit: enzymes in cluster α decreased their expression during the ripening, while enzymes in clusters β and γ increased their expression in mature fruits. The results suggest differential regulation for enzymes in different phylogenetic clusters suggesting evolutionary divergences in function (Perry et al 2020). In order to corroborate the transcriptomic results, expression analysis using qRT-PCR were performed. qRT-PCR expression analysis of genes belonging to the cluster α: Solyc12g0558840, Solyc02080660 and Solyc01g005830 showed a similar trend as the expression analysis from microarray data . The expression of these genes is decreasing during the fruit development. Contrary, the expression of the genes Solyc11g068440 and Solyc04g016470, belonging to the clade γ, are increasing during the development. Based on these results, functional analysis of these genes was designed. Vectors with the gene Solyc12g0558840 and Solyc11g068440 under ubiquitin constitutive promotor were performed. Experiment is still in process.
Dissemination & exploitation was a fundamental part of the fellowship in order to contribute socially and economically to Europe. However, most of the conferences and workshops expected during the last year were cancel or postponed. Alternatively, the beneficiary was attended to electronic conferences as 1st International Electronic Conference on Plant Science. And this summer will attend to the 7th International Conference on Plant Cell Wall Biology https://www.pcwb2021.com/(si apre in una nuova finestra) and Results from Marie Curie fellow will be presented.
A virtual format “ Be Curious 2020” was attended and participated in order to divulge science to the general public . outreach activities(si apre in una nuova finestra).
Currently there is a publication of the project: Louisa Perry, Yoselin Benitez-Alfonso and Candelas Paniagua “An In-Silico Approach to Identify Ripening Related beta-1,3- Glucanases and Their Role in Tomato Fruit” in the open access journal Proceedings.
Callmechanics was only developed for 6 months, for this reason the objectives of the project were not finished. The vectors that have been produced by the fellow will be used to carry on the rest of the objectives even after this fellowship is terminated. Strong collaboration between Yoselin Benitez-Alfonso´s group and José Mercado´s group has been established in order to finish the main goal of the project. The generated vectors will be a wonderful tool for both groups to study the effect on the fruit quality and shelf-life.
We would like to obtain plants (with the new collaboration University of Malaga-University of Leeds) producing fruits that maintain their firmness for a longer period of time, thus with reduced susceptibility to mechanical damage and pathogen attack during the postharvest period. This achievement will have a positive impact on the European Union economy by optimizing processes such as the frequency of harvesting, the handling and the transport procedures. It will also impact on human health and food security by reducing losses and the spreading of pathogens that thrive in mature soft fruits.
We would like to obtain plants (with the new collaboration University of Malaga-University of Leeds) producing fruits that maintain their firmness for a longer period of time, thus with reduced susceptibility to mechanical damage and pathogen attack during the postharvest period. This achievement will have a positive impact on the European Union economy by optimizing processes such as the frequency of harvesting, the handling and the transport procedures. It will also impact on human health and food security by reducing losses and the spreading of pathogens that thrive in mature soft fruits.