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      Azacytidine arrests ripening in cultivated strawberry ( Fragaria × ananassa) by repressing key genes and altering hormone contents

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          Abstract

          Background

          Strawberry ripening involves a number of irreversible biochemical reactions that cause sensory changes through accumulation of sugars, acids and other compounds responsible for fruit color and flavor. The process, which is strongly dependent on methylation marks in other fruits such as tomatoes and oranges, is highly controlled and coordinated in strawberry.

          Results

          Repeated injections of the hypomethylating compound 5-azacytidine (AZA) into green and unripe Fragaria × ananassa receptacles fully arrested the ripening of the fruit. The process, however, was reversible since treated fruit parts reached full maturity within a few days after AZA treatment was stopped. Transcriptomic analyses showed that key genes responsible for the biosynthesis of anthocyanins, phenylpropanoids, and hormones such as abscisic acid (ABA) were affected by the AZA treatment. In fact, AZA downregulated genes associated with ABA biosynthetic genes but upregulated genes associated with its degradation. AZA treatment additionally downregulated a number of essential transcription factors associated with the regulation and control of ripening. Metabolic analyses revealed a marked imbalance in hormone levels, with treated parts accumulating auxins, gibberellins and ABA degradation products, as well as metabolites associated with unripe fruits.

          Conclusions

          AZA completely halted strawberry ripening by altering the hormone balance, and the expression of genes involves in hormone biosynthesis and degradation processes. These results contradict those previously obtained in other climacteric and fleshly fruits, where AZA led to premature ripening. In any case, our results suggests that the strawberry ripening process is governed by methylation marks.

          Supplementary Information

          The online version contains supplementary material available at 10.1186/s12870-022-03670-1.

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          Most cited references58

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          Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening.

          Ripening of tomato fruits is triggered by the plant hormone ethylene, but its effect is restricted by an unknown developmental cue to mature fruits containing viable seeds. To determine whether this cue involves epigenetic remodeling, we expose tomatoes to the methyltransferase inhibitor 5-azacytidine and find that they ripen prematurely. We performed whole-genome bisulfite sequencing on fruit in four stages of development, from immature to ripe. We identified 52,095 differentially methylated regions (representing 1% of the genome) in the 90% of the genome covered by our analysis. Furthermore, binding sites for RIN, one of the main ripening transcription factors, are frequently localized in the demethylated regions of the promoters of numerous ripening genes, and binding occurs in concert with demethylation. Our data show that the epigenome is not static during development and may have been selected to ensure the fidelity of developmental processes such as ripening. Crop-improvement strategies could benefit by taking into account not only DNA sequence variation among plant lines, but also the information encoded in the epigenome.
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            Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine.

            The cytosine analogues 5-azacytosine (azacytidine) and 2'-deoxy-5-azacytidine (decitabine) are the currently most advanced drugs for epigenetic cancer therapies. These compounds function as DNA methyltransferase inhibitors and have shown substantial potency in reactivating epigenetically silenced tumor suppressor genes in vitro. However, it has been difficult to define the mode of action of these drugs in patients and it appears that clinical responses are influenced both by epigenetic alterations and by apoptosis induction. To maximize the clinical efficacy of azacytidine and decitabine it will be important to understand the molecular changes induced by these drugs. In this review, we examine the pharmacological properties of azanucleosides and their interactions with various cellular pathways. Because azacytidine and decitabine are prodrugs, an understanding of the cellular mechanisms mediating transmembrane transport and metabolic activation will be critically important for optimizing patient responses. We also discuss the mechanism of DNA methyltransferase inhibition and emphasize the need for the identification of predictive biomarkers for the further advancement of epigenetic therapies. (c) 2008 Wiley-Liss, Inc.
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              Abscisic acid plays an important role in the regulation of strawberry fruit ripening.

              The plant hormone abscisic acid (ABA) has been suggested to play a role in fruit development, but supporting genetic evidence has been lacking. Here, we report that ABA promotes strawberry (Fragaria ananassa) fruit ripening. Using a newly established Tobacco rattle virus-induced gene silencing technique in strawberry fruit, the expression of a 9-cis-epoxycarotenoid dioxygenase gene (FaNCED1), which is key to ABA biosynthesis, was down-regulated, resulting in a significant decrease in ABA levels and uncolored fruits. Interestingly, a similar uncolored phenotype was observed in the transgenic RNA interference (RNAi) fruits, in which the expression of a putative ABA receptor gene encoding the magnesium chelatase H subunit (FaCHLH/ABAR) was down-regulated by virus-induced gene silencing. More importantly, the uncolored phenotype of the FaNCED1-down-regulated RNAi fruits could be rescued by exogenous ABA, but the ABA treatment could not reverse the uncolored phenotype of the FaCHLH/ABAR-down-regulated RNAi fruits. We observed that down-regulation of the FaCHLH/ABAR gene in the RNAi fruit altered both ABA levels and sugar content as well as a set of ABA- and/or sugar-responsive genes. Additionally, we showed that exogenous sugars, particularly sucrose, can significantly promote ripening while stimulating ABA accumulation. These data provide evidence that ABA is a signal molecule that promotes strawberry ripening and that the putative ABA receptor, FaCHLH/ABAR, is a positive regulator of ripening in response to ABA.
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                Author and article information

                Contributors
                rivas@mpimp-golm.mpg.de
                bb1mublj@uco.es
                arfranco@uco.es
                Journal
                BMC Plant Biol
                BMC Plant Biol
                BMC Plant Biology
                BioMed Central (London )
                1471-2229
                7 June 2022
                7 June 2022
                2022
                : 22
                : 278
                Affiliations
                [1 ]GRID grid.411901.c, ISNI 0000 0001 2183 9102, Department of Biochemistry and Molecular Biology, , University of Cordoba, ; Edificio Severo Ochoa, Campus de Rabanales, E-14014 Córdoba, Spain
                [2 ]GRID grid.418390.7, ISNI 0000 0004 0491 976X, Max-Planck-Institute of Molecular Plant Physiology, ; Am Mühlenberg 1, 14476 Potsdam-Golm, Germany
                [3 ]GRID grid.510916.a, ISNI 0000 0004 9334 5103, Center of Plant Systems Biology and Biotechnology, ; Ruski Blvd. 139, 4000 Plovdiv, Bulgaria
                Article
                3670
                10.1186/s12870-022-03670-1
                9172142
                35672704
                be0075f0-c037-4da1-a7da-26758fd4a4a3
                © The Author(s) 2022

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

                History
                : 7 March 2022
                : 31 May 2022
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100004837, Ministerio de Ciencia e Innovación;
                Award ID: AGL2014-55784-C2-2-R
                Funded by: Ministerio de Economía, Industria y Competitividad
                Award ID: AGL2017-86531-C2-2-R
                Funded by: Ministerio de Economia y Competitividad
                Award ID: RYC-2014-15111
                Funded by: FundRef http://dx.doi.org/10.13039/501100011011, Junta de Andalucía;
                Award ID: Plan PAIDI2020
                Funded by: European Union's Horizon 2020
                Award ID: SGA-CSA No. 739582 under FPA No. 664620
                Funded by: Max Planck Institute of Molecular Plant Physiology (2)
                Categories
                Research
                Custom metadata
                © The Author(s) 2022

                Plant science & Botany
                azacytidine,demethylation,ripening,m5-cytosine,abscisic acid
                Plant science & Botany
                azacytidine, demethylation, ripening, m5-cytosine, abscisic acid

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