Epigenetic regulation in tomato fruit ripening

Fruit ripening is a crucial stage in quality development, influenced by a diverse array of internal and external factors. Among these factors, epigenetic regulation holds significant importance and has garnered substantial research attention in recent years. Here, this review aims to discuss the breakthrough in epigenetic regulation of tomato (Solanum lycopersicum) fruit ripening, including DNA methylation, N6-Methyladenosine mRNA modification, histone demethylation/deacetylation, and non-coding RNA. Through this brief review, we seek to enhance our understanding of the regulatory mechanisms governing tomato fruit ripening, while providing fresh insights for the precise modulation of these mechanisms.


Introduction
As a unique organ of angiosperms, fruit provides rich dietary fiber, vitamins and other nutrients for human beings, and is an important component of healthy dietary structure (Giovannoni, 2001;Chen et al., 2020).Fruit ripening is a key period for the formation of fruit edible quality, which is a complex developmental process involves changes in fruit texture, pigment accumulation, formation of aroma and flavor substances, reduction of resistance and other traits, and regulated by many internal and external factors (Giovannoni, 2004;Ji and Wang, 2023).The internal factors mainly include transcription factors and hormones, while the external factors mainly include various biological factors and abiotic factors.According to the different respiratory patterns, fruits can be divided into two types: climacteric and non-climacteric (Mcmurchie et al., 1972).During the ripening process, the respiratory intensity and ethylene release of climacteric fruits appeared the accompanied burst, such as tomato, apple and banana.However, there was no significant change in respiratory intensity and ethylene release in non-climacteric fruits, such as strawberry, grape, citrus (Shinozaki et al., 2018).Two systems of ethylene biosynthesis (System I and System II) play vital roles in the development and ripening of climacteric fruits.Immature fruits and other organs of the plant continuously produce low concentrations of ethylene, that is, the background concentration of ethylene.System I ethylene regulates ethylene synthesis of background concentration in a negative feedback way and participates in fruit development, while system II ethylene is produced in an autocatalytic manner during fruit ripening (Liu M. et al., 2015).The consensus view asserts that ethylene usually collaborates with transcription factors to collectively orchestrate the regulation of fruit ripening (Li et al., 2022).In tomato, MADS-box transcription factors are recognized as key transcriptional regulators that govern fruit ripening (Smaczniak et al., 2012;Gapper et al., 2013).Among these, the MADS-box transcription factor RIPENING-INHIBITOR (RIN) plays a crucial role in the regulation of tomato fruit ripening and is considered a master regulator (Vrebalov et al., 2002;Li et al., 2019).Researchers have employed Chromatin Immunoprecipitation (ChIP) techniques to progressively identify a series of target genes regulated by RIN.These target genes are involved in various aspects of fruit ripening, including ethylene biosynthesis and signaling (Ito et al., 2008;Li et al., 2011), fruit softening (Fujisawa et al., 2011), pigment synthesis (Martel et al., 2011), aroma compound synthesis (Qin et al., 2012), protein ubiquitination (Wang et al., 2014), and sucrose metabolism (Qin et al., 2016).TOMATO AGAMOUS-LIKE1 (TAGL1) is another member of the MADS box family.In tomato, silencing of TAGL1 inhibits the fruit ripening process, leading to thinner fruit skin, reduced ethylene release, and an incomplete transition to red coloration (Vrebalov et al., 2009).It was reported that TAGL1 can regulate ethylene biosynthesis by activating the expression of the ACS2 gene (Itkin et al., 2009).FRUITFULL1 (FUL1) and FUL2 are also members of the MADS box family of transcription factors, and both proteins can interact with RIN (Bemer et al., 2012).Simultaneous silencing of both genes led to a reduction in tomato lycopene content (Bemer et al., 2012).FUL1/FUL2 target over 800 genes, with some overlapping with RIN target genes.In vitro experiments have indicated that FUL1, FUL2, RIN, and TAGL1 may form a quaternary complex to collectively regulate fruit ripening (Bemer et al., 2012;Fujisawa et al., 2014).In the context of tomato fruit, RIN and TAGL1 form a complex that triggers the activation of ethylene biosynthesis genes, thereby establishing a positive feedback loop that generates autocatalytic ethylene (Lü et al., 2018;Ji et al., 2020).This MADS-type circuit subsequently stimulates downstream ripening-associated genes.
Tomato (Solanum lycopersicum), an important economic crop, is cherished for its delicious and nutritious fruit.With a relatively small genome size (~900 Mb), well-defined genetic background, abundant mutant resources, and ease of genetic transformation, the tomato has emerged as a model plant for studying the biology of climacteric fruit development (Giovannoni et al., 2017;Liu et al., 2022).Additionally, the distinct stages of fruit ripening, coupled with its evident attributes, make it an ideal candidate for investigating the biology of climacteric fresh fruit ripening.A large number of studies have focused on transcription and ethylene regulation of tomato fruit ripening.Due to the rapid development of new sequencing technologies, the study of epigenetics has greatly promoted the molecular mechanism of tomato fruit ripening (Giovannoni et al., 2017).An increasing body of evidence suggests that epigenetic mechanisms play a role in modulating the process of tomato fruit ripening, and the regulatory mechanisms of some important proteins and genes have been analyzed (Zhong et al., 2013;Giovannoni et al., 2017;Liu et al., 2022).Epigenetics refers to heritable changes in gene expression without altering the original gene sequence (Eichten et al., 2014;Ji and Wang, 2023).Epigenetic regulation encompasses diverse mechanisms, including methylation modification of DNA or RNA, histone modification, non-coding RNA.Here, this review provides an overview of the breakthrough and future prospects for studies of epigenetic regulation in tomato fruit ripening.
2 Epigenetic regulation of tomato fruit ripening 2.1 DNA/RNA methylation DNA methylation referred a conserved process involving the transfer of a −CH3 group to the fifth carbon of cytosine residues, resulting in the formation of 5-methylcytosine (5mC), exerting a significant impact on chromatin function, usually down-regulating gene expression (Zhang et al., 2018;He et al., 2022).A classic example of methylation-mediated regulation in plant fruit ripening arises from investigations of the tomato cnr (Colorless non-ripening) mutants.CNR was located on tomato chromosome 2, belonged to the SBP (squamosa promoter binding protein) family of transcription factors.Compared with the wild type, ethylene synthesis, fruit softening and carotenoid synthesis of cnr mutant fruits were inhibited (Eriksson et al., 2004).At the same time, the fruit texture of the mutant was loose, the intercellular adhesion was decreased, and the fruit was easy to crack.It was found that CNR DNA sequence was not changed in cnr mutants, while the promoter region showed hypermethylation, and CNR transcription was inhibited (Manning et al., 2006).Nonetheless, recent studies have indicated that following the application of CRISPR/Cas9 gene editing technology to knock out CNR, there was no substantial alteration observed in the fruit ripening phenotype in comparison to the wild type.Furthermore, the fruit phenotype associated with the cnr mutant could not be replicated (Gao et al., 2019).Therefore, the molecular regulation mechanism of this transcription factor still needs further study.
To determine the intrinsic link between DNA methylation and fruit ripening, researchers treated tomato fruits with methyltransferase inhibitor 5-azacytidine and found that the ripening process was accelerated, while hypermethylation was observed in the cnr and rin (ripening inhibitor) mutants fruits (Zhong et al., 2013).Further genome-wide methylation sequencing found that DNA methylation levels decreased continuously as the fruit matured (Zhong et al., 2013).The results indicated that DNA methylation was a dynamic process during different fruit ripening stages.Functional analysis of four DNA DEMETER-like DNA demethylases (DMLs) in tomato showed that SlDML2 was highly expressed in fruit and showed ripening related expression patterns (Liu R. et al., 2015).After this gene was silenced or knocked out, tomato fruit ripening process was delayed, indicating that DNA demethylase played an important role in the regulation of fruit ripening (Liu R. et al., 2015;Lang et al., 2017).A recent study found that SlDML2 not only regulated RIN expression, but also regulated RIN binding in tomato fruit ripening process (Niu et al., 2022).In addition, studies have shown that RNA demethylase Solanum lycopersicum AlkB homolog 2 (SlALKBH2) can bind the mRNA of SlDML2 and regulate the mRNA stability of SlDML2 through N 6 -Methyladenosine (m 6 A) demethylation (Zhou et al., 2019).After SlALKBH2 was knocked out by CRISPR/Cas9 gene editing technology, the fruit ripening process was delayed.These results indicate that there is an intrinsic relationship between RNA methylation and DNA methylation.There is also evidence indicating the participation of DNA methyltransferases in the regulation of tomato fruit ripening.When knocked out Solanum lycopersicum methyltransferase1 (SlMET1), most ripening-related RIN target genes were up-regulated, indicating that SlMET1 may repress these genes (Yang et al., 2019).These findings suggest that DNA methylation plays an important role, not in isolation, but in synergy with other signals to regulate tomato ripening (Figure 1).
Currently, our understanding of the various signaling molecules involved in DNA methylation is not comprehensive enough.Future studies are needed to further enrich this molecular regulatory network.

Histone modification
Histone modification refers to the methylation, acetylation, phosphorylation, and other modifications of the amino terminus of histones (Patel and Wang, 2013).These modifications can alter the state of chromatin and regulate gene expression.Up to now, 52 histone methyltransferases (HMTs), 26 histone demethylases The regulation network of DNA methylation, RNA methylation, histone modification and non-coding RNA in tomato fruit ripening.SlALKBH2 regulates the stability of SlDML2 RNA by RNA m 6 A demethylation, enhancing its stability.Conversely, SlDML2 promotes the expression of SlALKBH2 through DNA demethylation, ultimately affecting fruit ripening.The histone demethylase SlJMJ6 eliminates H3K27me marks from SlDML2, RIN and ripening-associated genes, consequently promoting fruit ripening.On the other hand, histone demethylase SlJMJ7 erases H3K4me modifications from these genes, thus contributing to the inhibition of fruit ripening.Tomato methyltransferase SlMET1, Histone deacetylases SlHDA1, SlHDA3 and SlHDT1 may negatively regulate ripening-associated genes to control fruit ripening, while SlHDT3 exhibits opposing regulatory effects.PRC1 protein SlLHP1b could bind H3K27me mark in regions of ripening-associated chromatin, targeting ripening-related genes, repressing fruit ripening.Meanwhile, SlLHP1b interacted with PRC2 protein SlMSI1, negatively regulating tomato fruit ripening.The microRNA gene SlMIR164A was involved in negative regulation of tomato fruit ripening.
(HDMs), 32 histone acetyltransferases (HATs), and 15 histone deacetylases (HDACs) have been identified in tomato genome (Aiese Cigliano et al., 2013).Relevant investigations have demonstrated the significant contribution of histone methylation to fruit ripening.Plant development is profoundly shaped by the pivotal functions executed by Polycomb group (PcG) proteins, which epigenetically suppress the transcription of target genes.Overexpression of SlMSI1 (Solanum lycopersicum MULTICOPY SUPPRESSOR OF IRA1), a component of polycomb repressive complex 2 (PRC2), in the histone H3K27 methylase protein complex in tomato inhibited the expression of ripening related genes and hindered the fruit ripening process (Liu et al., 2016).Similar results were found in PRC1 protein SlLHP1b (Solanum lycopersicum Like Heterochromatin Protein 1b).SlLHP1b could bind H3K27me mark in regions of ripening-associated chromatin, targeting ripening-related ethylene biosynthesis, carotenoid biosynthesis and RIN targeted genes, repressing fruit ripening (Liang et al., 2020).Meanwhile, SlLHP1b interacted with SlMSI1, suggesting a potential collaborative role of these two proteins in regulating tomato fruit ripening (Liang et al., 2020).Enhancer of Zeste (EZ) Polycomb group protein SlEZ2 was a member of PRC2, reduction of this gene expression resulted in alteration in fruit development and ripening (Boureau et al., 2016).Moreover, genome-wide analysis of H3K27e3 revealed that this epigenetic modification was negatively associated with ripeningrelated genes expression (Lü et al., 2018).SlJMJ6 (Solanum lycopersicum Jumonji C domain-containing protein 6) encodes a histone lysine H3K27 demethylase, and the overexpression of SlJMJ6 accelerated the process of fruit ripening (Li et al., 2020).Subsequent investigations revealed that SlJMJ6 could directly target 32 genes, including RIN, SlDML2, thereby modulating the transcription of these genes.Another study showed that H3K4 demethylase SlJMJ7 modulated tomato fruit ripening by regulating ethylene biosynthesis genes, ripening related transcription factor and DNA demethylation genes expression through H3K4me3 demethylation (Ding et al., 2022).This finding revealed a crosstalk between histone methylation and DNA methylation.
Apart from histone methylation modifications, histone acetylation modifications also participate in the regulation of tomato fruit ripening.It has been discovered that distinct histone deacetylases play varying roles in the regulation of fruit ripening.Silencing of SlHDA1 (Solanum lycopersicum histone deacetylase1), SlHDA3 and SlHDT1 (Solanum lycopersicum HD-tuins1) accelerated fruit ripening, while the silencing of SlHDT3 slowed fruit ripening (Guo et al., 2017a;Guo et al., 2017b;Guo et al., 2018;Guo, 2022).In addition, gene knockout experiment revealed that the histone variant Sl_H2A.Z regulated the expression of carotenoids biosynthesis related genes, including SlPSY1 (Solanum lycopersicum PHYTOENE SYNTHASE 1), SlPDS (Solanum lycopersicum PHYTOENE DESATURASE 1), and SlVDE (Solanum lycopersicum VIOLAXANTHIN DE-EPOXIDASE) in tomato ripening (Yang et al., 2021).These findings indicate that histone modifications may play a significant regulatory role in tomato fruit ripening.Future research will focus on further identifying the histone modification molecules involved in the regulation of ripening.

Non-coding RNA
In addition to DNA/RNA methylation and histone modification, non-coding RNA also participates in the regulation of tomato fruit ripening.Non-coding RNAs are a class of RNAs that do not code for proteins (Ma et al., 2020).In studies on regulation of fruit ripening, researchers mainly focus on microRNA (miRNA), long non-coding RNA (lncRNA) and circular RNA (circRNA).Researchers performed high-throughput sequencing on tomato fruits and identified a collection of non-coding RNAs that could potentially participate in fruit ripening (Gao et al., 2015;Zhu et al., 2015;Tan et al., 2017).It was found that the expression of 19 conserved microRNAs in fruits of rin mutants was significantly different from that of wild types (Gao et al., 2015).Another study analyzed lncRNAs in rin mutants and wild-type fruits, and identified 677 differentially expressed lncRNAs (Zhu et al., 2015).Some identified lncRNAs were silenced instantaneously using virusinduced gene silencing technology, leading to a delay in the fruit ripening process.A total of 1018 circRNAs were identified in tomato fruits through deep sequencing of RNA samples, with rRNA and RNase R digestion removed.Notably, some of these circRNAs showed a close correlation with pigment synthesis (Tan et al., 2017).Furthermore, a recent review has synthesized the current findings regarding non-coding RNAs associated with fruit ripening.This review delineated that out of these non-coding RNAs, 40 were linked to the ethylene pathway, 8 to color, 14 to flavor, and 32 to texture in tomato fruit ripening (Ma et al., 2020).In terms of the specific roles of individual miRNA genes, it was reported that upon the knockout of SlMIR164A using CRISPR/Cas9 gene editing technology, the fruit ripening process accelerated, and there were alterations in sugar and organic acid contents (Lin et al., 2022).It is evident that non-coding RNAs may have crucial functions in the process of tomato fruit ripening.With further functional verification of non-coding RNAs, researchers will gain a deeper understanding and be able to analyze the mechanisms by which these non-coding RNAs regulate fruit ripening.

Discussion
The functional analysis of the key components of DNA methylation, RNA methylation, histone modification and noncoding RNA in tomato fruit ripening regulation revealed a complex regulation network (Figure 1).Previous study showed that the RNA demethylase SlALKBH2 could regulate the mRNA stability of the DNA demethylase SlDML2 through m 6 A demethylation (Zhou et al., 2019), elucidating the interaction between RNA methylation and DNA methylation in tomato fruit ripening regulation.Meanwhile, the H3K4 demethylase SlJMJ7 could repress the DNA demethylation gene SlDML2 expression by H3K4me3 demethylation, thus establishing a crosstalk between histone and DNA demethylation in tomato fruit ripening regulation (Ding et al., 2022).An integrated assessment of alterations in genome methylation, long non-coding RNAs, circular RNAs, micro RNAs, and fruit metabolites unveiled numerous differentially expressed genes (DEGs).These DEGs encompass differentially methylated regions that encode transcription factors and pivotal enzymes linked to ethylene or carotenoid pathways, which could potentially be targeted by differentially expressed noncoding RNAs (Zuo et al., 2020).Existing research indicates that tomato fruit ripening regulatory factors do not act in isolation but rather function in coordination.Currently, our understanding of these cooperative mechanisms is not sufficiently deep.Future research can focus on exploring the interactions among DNA or RNA methylation modifications, histone modification, and noncoding RNA, which will give a more comprehensive and intriguing regulatory network for tomato fruit ripening.At present, research efforts are predominantly focused on DNA demethylases, RNA demethylases, histone demethylases, and histone deacetylases.There is relatively less research conducted on DNA methyltransferases, RNA methyltransferases, histone methyltransferases, and histone acetyltransferases.In the future, there should be a strengthening of research in these areas to gain a more comprehensive understanding.
Currently, the online Tomato Epigenome Database (http:// ted.bti.cornell.edu/epigenome/)offers a valuable resource for searching DNA methylation patterns and accessing information on cytosine methylation, gene expression, small RNA, and RINbinding profiles through the Genome Browser.Further development of similar online tools and standalone software is essential to facilitate rapid and convenient investigations into the role of epigenetics in tomato fruit ripening.Additionally, advancements in sequencing and experimental technologies, such as 3D genome analysis, single-cell sequencing, and spatial-temporal omics, will enable a more extensive and in-depth exploration of the epigenetic regulation within the tomato fruit ripening regulatory network in the future.