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Transcriptional RegulAtory Network controlling Strawberry Fruit Ripening and Quality

Periodic Reporting for period 4 - TRANSFR-Q (Transcriptional RegulAtory Network controlling Strawberry Fruit Ripening and Quality)

Reporting period: 2019-12-01 to 2021-11-30

Ripening is the critical step for the development of flavour quality in fruit. This character has significantly declined in many fleshy fruits over recent decades, primarily due to the focus of current breeding programs on agronomic traits such as production, firmness, and postharvest shelf life. This strategy has caused a tunnelling effect on genetic variability in many crops. This is particularly significant in strawberry, where current cultivars are derived from a narrow germplasm stock. Moreover, strawberry fruits possess two notable features: i) the fleshy part of the strawberry fruit is the result of the enlargement of the flower receptacle, while the true fruits are the dry structures called achenes, anchored on the surface of the receptacle; ii) while the hormone ethylene is essential for the ripening of climacteric fruits such as tomato, ethylene only plays a marginal role in strawberry fruits, and as such is considered non-climacteric. Therefore, improving fruit flavour in present strawberry varieties requires two important breakthroughs: 1) a precise understanding of non-climacteric and receptacle ripening regulation that will allow the targeting of relevant quality genes, and 2) the identification of unexploited allelic variants from wild germplasm to be introgressed through the generation of novel breeding lines. In our project, we are interested in generating information and tools that will help to achieve these goals. Specifically, we have followed three specific aims in this project:
1) To characterize molecularly the strawberry fruit ripening performing transcriptomics and metabolomics analyses
2) To identify and investigate the role played by key transcription factors (TFs) regulating fruit ripening
3) To uncover the gene regulatory network for these key TFs, and to study their impact on fruit quality characters
4) To identify novel genes controlling fruit quality characters of the ripe fruit using a germplasm collection of woodland strawberry (Fragaria vesca) accessions.
Overall, TRANSFR-Q project final aim is to gain deep knowledge in the molecular processes controlling strawberry fruit ripening, and identifying genes with a key role in the acquisition of quality characters in order to transfer this knowledge into new and improved breeding lines. Moreover, we plan to establish comprehensive genomic resources not only for strawberry but also for the Rosaceae community, an aspect of utmost importance since this family contains many economically important fruit trees, including apple, peach, cherry, apricot and almond.
During this project we have performed the following main tasks:
1) We have studied the global gene expression and the metabolome (primary and secondary metabolites) in different ripening stages of the woodland strawberry Fragaria vesca fruit. Analysis of the transcriptome allowed us to identify and characterize transcription factors (TFs) that play essential roles regulating the ripening process. Among others, we have identified FaRIF as one of the more upstream regulators of strawberry ripening described so far (Martín-Pizarro, Plant Cell, 33:1574-1593, 2021).
2) We also performed a tissue- and stage-specific transcriptomics study of strawberry receptacles. Using both transcriptomics datasets, we generated Gene Regulatory Networks combining spatial and temporal data that allowed us to identify TFs with a role in specific tissues and stages of the fruit. We have characterized the role of some of these TFs so far, in particular those expressed in the external layer of ripe receptacles. Our data suggest that these genes are good candidates to play important roles regulating fruit color and the resistance to abiotic and biotic stresses, having therefore a great biotechnological interest.
3) We optimized the genome edition system CRISPR/Cas9 in the octoploid strawberry F. x ananassa. We successfully edited the TM6 gene, not only validating this approach but also deciphering the role of this gene in strawberry for the first time (Martín-Pizarro, J Exp Bot, 70:885-895, 2019). This work positions CRISPR/Cas9 as a feasible approach for gene functional analysis in strawberry despite its high ploidy level, becoming a good alternative to the sometimes-inefficient post-transcriptional gene silencing approaches.
4) We have resequenced the genome and characterised for fruit quality a F. vesca germplasm collection, providing a genomic dataset resource of great importance for the community. Furthermore, a GWAS approach has allowed us to identify genes involved in the biosynthesis of a number of volatile compounds with an important role in the fruit aroma, a key character for fruit quality.

Some of this work has been already published in high-impact peer-reviewed journals (Plant Cell, Journal of Experimental Botany, Plant Biotechnology Journal, etc), but most of it is currently in preparation for publication. The published work has been widely disseminated in many scientific national and international conferences and outreach events, as well as in press and social media.
The central aspect of our project is dedicated to understanding how fruit ripening is controlled in strawberry fruits and identifying genes involved in fruit quality in order to apply this knowledge to new breeding programs. In order to accomplish these objectives, I will summarize the progress achieved so far, and the results we expect until the end of the project:
1) We have characterized and integrated the global gene expression and metabolic changes that occur during strawberry fruit ripening, and moreover, we have obtained a tissue-specific transcriptome. The latter analysis provides a much more detailed gene expression atlas for strawberry fruits than any transcriptomic study performed so far.
2) We have identified TFs that control the ripening process of the strawberry fruit, characterizing their role using molecular, genetic, genomic and metabolomic approaches and gaining, for the first time, a deep understanding of the gene regulatory pathways leading to strawberry fruit ripening. Furthermore, we have identified the direct targets of these key TFs, an approach that is still not commonly applied to this species.
3) We were pioneers in the use of the genome-editing tool CRISPR/Cas9 in the commercial octoploid strawberry plants. This methodology will be of great value, not only to the scientific community interested in this species but also to strawberry breeding companies.
4) Finally, we have used the underexploited genetic diversity present in the woodland species Fragaria vesca and identified genes involved in fruit quality traits, opening the generation of new breeding lines up to the strawberry industry in the future.

In summary, TRANSFR-Q has aimed to uncover key regulatory genes that govern the transcriptional networks leading to the quality characters of strawberry fruits. In addition to providing an essential understanding of fruit ripening, it will set the basis for breeding quality characters in current strawberry cultivars in the future.
Representative image of the project