Periodic Reporting for period 1 - ARCATOM (Exploring the role of the mitochondrial alternative respiration in carotenoid biosynthesis during tomato fruit ripening)
Période du rapport: 2018-10-01 au 2020-09-30
Fruit ripening is a highly coordinated developmental process that facilitates animal-driven dispersal of mature seed. Tomato (Solanum lycopersicum) is the main model system for fruit ripening studies at biochemical, genetic and molecular levels. In this economically relevant climacteric fruit, a sudden increase in respiration takes place at the onset of ripening, usually in concert with increased production of ethylene that eventually impacts fruit color, firmness, taste, and flavor. Tomato ripening also involves the differentiation of chloroplasts into chromoplasts that accumulate high levels of β-carotene (pro-vitamin A) and lycopene, carotenoid pigments that provide the characteristic orange and red colors to the ripe fruit. Carotenoids also act as precursors for the synthesis of volatiles that contribute to the characteristic aroma of tomato fruit. Furthermore, dietary carotenoids are powerful health-promoting antioxidants. While a respiratory process supplying ATP has been proposed to function in tomato fruit chromoplasts, it is likely that most ATP and metabolic precursors for plastidial metabolism are imported into chromoplasts. The scientific aim of ARCATOM was to address the specific contribution of mitochondrial respiration to the supply of energy and carbon for the production of carotenoids in tomato fruit during ripening.
The project provides novel information into the interactions between primary and secondary metabolism which are still poorly understood. Specifically, the role and regulation of the alternative respiration during fruit ripening, which is still a matter of debate, has been unravelled. The outcomes of this research can be used to design new biotechnological strategies in order to improve vegetable crops productivity and fruit quality.Therefore this research is aligned with the strategy of Horizon 2020 of the European Commission, in the “Food security, sustainable agriculture and forestry, marine and maritime and inland water research and the bioeconomy”.
The project provides novel information into the interactions between primary and secondary metabolism which are still poorly understood. Specifically, the role and regulation of the alternative respiration during fruit ripening, which is still a matter of debate, has been unravelled. The outcomes of this research can be used to design new biotechnological strategies in order to improve vegetable crops productivity and fruit quality.Therefore this research is aligned with the strategy of Horizon 2020 of the European Commission, in the “Food security, sustainable agriculture and forestry, marine and maritime and inland water research and the bioeconomy”.
In ARCATOM, we proposed to determine the in vivo AOP and COP respiration during fruit ripening (objective 1) in tomato wild-type (WT) plants and ghost mutants defective in chromoplast-associated respiration. In addition, we also proposed to explore the impact of changes in mitochondrial alternative respiration on primary and secondary metabolic pathways (objective 2), with a focus on carotenoids and other MEP-derived health-related compounds.
The first objective has been fully achieved. Firstly, the oxygen-isotope fractionation technique was set up for measuring the respiratory in vivo activities in tomato pericarp samples . This was particularly challenging but difficulties were overcome. The rather unique technique is now available to explore respiration and electron partitioning in fruits from tomato and also in other climacteric and non-climateric species, which opens fascinating opportunities for fruit metabolism research. After this set up, we present for the first time the increase on the in vivo activation of the AOX pathway during climacteric respiration. Thereafter, pericarp samples from WT and ghost mutant tomatoes were used to perform gene expression analysis of respiratory and carotenoid pathways. Finally, primary metabolites and carotenoid profiles were obtained in pericarp samples of wild-type and ghost fruit at different maturation stages. On the other hand, the second objective could be partly achieved. The generation of the genetically-modified tomato plants after the Agrobacterium-mediated transformation has been substantially delayed due to some technical issues and the subsequent entering into the period of the COVID-19 outbreak. Nevertheless, the recently obtained genetically-modified plant material will be used to pursue this research in the frame of my recently granted tenure-track position.
The results derived from this WP1 have been presented in three conferences as planned in the proposed measures to exploit and disseminate the action results. In this line, a manuscript including the outcomes of WP1 is going to be submitted soon to a first decile journal from Plant Sciences Category (WOS). In addition to the outcomes reported above, the researcher has been involved in two collaboration projects with the supervisor of the host group in close connection with the ARCATOM objectives on linking respiratory and carotenoid metabolism; one study has been published in PNAS (Llorente et al., 2020) and the other has been recently accepted in New Phytologist (Barja et al., 2021). Furthermore, the researcher has published two review publications, one as first (Florez-Sarasa et al., 2020) and another as co-author (Ortiz et al., 2020), on the general topic of ARCATOM (plant alternative respiration).With regard to training activities, the researcher has performed several courses/workshops and supervised students, all being very valuable for his future activities in research and transfer knowledge as an independent group leader. Also, the researcher has been part of the organization and scientific committee of a research conference for early career researchers (mainly postdocs and young PIs). Finally, the researcher has been substantially involved in outreach activities after participating in five different events, most of them being the responsible person.
The first objective has been fully achieved. Firstly, the oxygen-isotope fractionation technique was set up for measuring the respiratory in vivo activities in tomato pericarp samples . This was particularly challenging but difficulties were overcome. The rather unique technique is now available to explore respiration and electron partitioning in fruits from tomato and also in other climacteric and non-climateric species, which opens fascinating opportunities for fruit metabolism research. After this set up, we present for the first time the increase on the in vivo activation of the AOX pathway during climacteric respiration. Thereafter, pericarp samples from WT and ghost mutant tomatoes were used to perform gene expression analysis of respiratory and carotenoid pathways. Finally, primary metabolites and carotenoid profiles were obtained in pericarp samples of wild-type and ghost fruit at different maturation stages. On the other hand, the second objective could be partly achieved. The generation of the genetically-modified tomato plants after the Agrobacterium-mediated transformation has been substantially delayed due to some technical issues and the subsequent entering into the period of the COVID-19 outbreak. Nevertheless, the recently obtained genetically-modified plant material will be used to pursue this research in the frame of my recently granted tenure-track position.
The results derived from this WP1 have been presented in three conferences as planned in the proposed measures to exploit and disseminate the action results. In this line, a manuscript including the outcomes of WP1 is going to be submitted soon to a first decile journal from Plant Sciences Category (WOS). In addition to the outcomes reported above, the researcher has been involved in two collaboration projects with the supervisor of the host group in close connection with the ARCATOM objectives on linking respiratory and carotenoid metabolism; one study has been published in PNAS (Llorente et al., 2020) and the other has been recently accepted in New Phytologist (Barja et al., 2021). Furthermore, the researcher has published two review publications, one as first (Florez-Sarasa et al., 2020) and another as co-author (Ortiz et al., 2020), on the general topic of ARCATOM (plant alternative respiration).With regard to training activities, the researcher has performed several courses/workshops and supervised students, all being very valuable for his future activities in research and transfer knowledge as an independent group leader. Also, the researcher has been part of the organization and scientific committee of a research conference for early career researchers (mainly postdocs and young PIs). Finally, the researcher has been substantially involved in outreach activities after participating in five different events, most of them being the responsible person.
The technical set-ups and conclusions derived from the work carried out in ARCATOM open new and exciting opportunities for the research field on fruit energy metabolism. The oxygen-isotope fractionation technique was set up for measuring the in vivo activities of the two main respiratory in tomato pericarp samples by overcoming different technical difficulties that have been limiting the advance in this research field. We propose that a climacteric burst of non-phosphorylating respiration allows a high rate of glycolytic and TCA cycle fluxes to provide the carbon precursors for the synthesis of ethylene and carotenoids. Moreover, the responses of primary metabolism in ghost tomato fruits indicate a similar climacteric response in the absence of chromorespiration as compared to wild-type fruits, although with some re-arrangements in stress-related amino acid metabolism. Finally, the obtained constructs and respiratory-altered tomato lines will enable not only to pursue on the study about the role and regulation of respiration in fruit metabolism, but also in other non-photosynthetic (i.e. roots) and photosynthetic tissues.
ARCATOM project has contributed to the advance on the knowledge about the regulation of plant respiration which is crucial for designing new strategies for mitigating the effects of climate change in plants, as has been recently reviewed by the researcher (Florez-Sarasa et al., 2020). In this context, we envisage a possibility for applying the research outcomes of ARCATOM to breeding programs of important crops, such as tomato, for Spanish agriculture at the medium/long term. Therefore, this research is aligned with the societal challenges (SC) described in the Spanish National Plan for Scientific and Technical Research and Innovation (SNPSTRI) 2017-2020, particularly the SC2 (Bioeconomy) and partly with SC5 (Climate change). The proposal also fits in the strategy of Horizon 2020 of the European Commission, in the “Food security, sustainable agriculture and forestry, marine and maritime and inland water research and the bioeconomy”.
ARCATOM project has contributed to the advance on the knowledge about the regulation of plant respiration which is crucial for designing new strategies for mitigating the effects of climate change in plants, as has been recently reviewed by the researcher (Florez-Sarasa et al., 2020). In this context, we envisage a possibility for applying the research outcomes of ARCATOM to breeding programs of important crops, such as tomato, for Spanish agriculture at the medium/long term. Therefore, this research is aligned with the societal challenges (SC) described in the Spanish National Plan for Scientific and Technical Research and Innovation (SNPSTRI) 2017-2020, particularly the SC2 (Bioeconomy) and partly with SC5 (Climate change). The proposal also fits in the strategy of Horizon 2020 of the European Commission, in the “Food security, sustainable agriculture and forestry, marine and maritime and inland water research and the bioeconomy”.