In plants, L-ascorbic acid(AsA)is a multifunctional molecule that serves critical roles in development processes and stress response (Smirnoff, 2000; Arrigoni and Tullio, 2002; Barth et al., 2006; Gest et al., 2013). As a primary antioxidant, L-ascorbic acid keeps active oxygen under normal levels to protect plants in photosynthesis (Smirnoff, 2000). L-ascorbic acid is also involved in regulation of flowering, fruit development, senescence and response to biotic and abiotic stress (Conklin and Barth, 2004; Barth et al., 2006; Gallie, 2013). For human health, L-ascorbic acid provide beneficial effects, such as enhancing antioxidant and anticancer activities (Raiola et al., 2014; Macknight et al., 2017; Salehi et al., 2019). Unable to synthesize L-ascorbic acid, humans can only get L-ascorbic acid from plant-based foods, especially tomato. For this reason, L-ascorbic acid contributes to the major feature of fruit nutrition and antioxidant capacity (Law and Jacobsen, 2010).
In higher plants, major biosynthesis pathways of L-ascorbic acid have been clarified, including the D-mannose/L-galactose pathway, D-glucosone pathway, D-galacturonate and myo-inositol pathway (Wheeler et al., 1998; Lorence et al., 2004; Bulley and Laing, 2016). In tomato, multiple enzymes are involved in each biosynthesis pathway, including GDP-D-mannose pyrophosphorylase (GMP), GDP-D-mannose-3,5-epimerase (GME), GDP-L-galactose-phosphorylase (GGP), L-galactose-1-P phosphatase (GPP), L-galactose dehydrogenase (GalDH); L-galactono-1,4-lactone dehydrogenase (GLDH), Myo-inositol oxygenase (MIOX) and D-galacturonate reductase (GalUR) (Smirnoff and Wheeler, 2000; Davey et al., 2006; Zou et al., 2006; Mellidou and Kanellis, 2017; Munir et al., 2020). In plants, reduced L-ascorbic acid can be oxidized by ascorbate peroxidase (APX) and ascorbate oxidase (AO) (Bulley and Laing, 2016). Moreover, L-ascorbic acid accumulation also depends on conversion between oxidized and reduced L-ascorbic acid through the AsA-GSH cycle involving monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) (Chen et al., 2003; Gallie, 2013; Mellidou and Kanellis, 2017).
In recent years, several transcription factors and proteins have been reported in regulation of L-ascorbic acid biosynthesis (Wang et al., 2013; Mellidou and Kanellis, 2017). Some of these genes enhancing AsA accumulation through positively regulating the transcription of AsA biosynthesis genes. For instance, overexpression of AtERF98 increases AsA level by directly regulation expression of AsA synthesis genes in the D-Man/L-Gal pathway while the knockout mutant erf98-1 displayed decreased AsA contents (Zhang et al., 2012). In Arabidopsis, mutation of KONJAC1 (KJC1) and KJC2 cause enhanced activity of VTC1 and GMP (Sawake et al., 2015). In tomato, two transcription factors, SlHZ24 and SlbHLH59, promote AsA accumulation via binding to promoters of AsA biosynthesis genes such as GDP-D-mannose pyrophosphorylase 3 (SlGMP3) (Hu et al., 2016; Ye et al., 2019). Nevertheless, others play negative roles on AsA biosynthesis. In Arabidopsis, AMR1 negatively affects expression of multiple genes encoding enzymes of the Man/l-Gal pathway, resulting in reduced AsA levels (Zhang et al., 2009). In tomato, SlNFYA10 modulates AsA biosynthesis through directly reducing expression of SlGME1 and SlGGP1 (Chen et al., 2020). Interaction of AtVTC1 and AtCSN5B causes ubiquitination-dependent AtVTC1 degradation with low AsA levels (Wang et al., 2013).
DNA methylation is a major form of epigenetic variations in plants and animals. This epigenetic modification of DNA occurs at the 5’ position of cytosine in symmetric sequence contexts (CG and CHG) and asymmetric sequence context (CHH) (Bender, 2004; He et al., 2011; Zhang and Zhu, 2012). In plants, DNA methylation contributes to many biological processes through altering gene expression and genome stability, such as fruit ripening, seed germination and stress response (Goll and Bestor, 2005; Dowen et al., 2012; Zhang et al., 2018; Liu and Lang, 2020). Establish of de novo DNA methylation is catalyzed by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) (Cao et al., 2003; Matzke and Mosher, 2014). CG and CHG methylation are maintained by METHYLTRANSFERASE 1 (MET1) and CHROMOMETHYLASE 3 (CMT3) respectively (Law and Jacobsen, 2010; Bewick et al., 2017). Over the years, DNA demethylation has been verified in developmental processes based on DNA demethylases, including Repressor of Silencing 1 (AtROS1) and DEMETER-LIKE 2 (DML2) (Lei et al., 2015; Tang et al., 2016; Liu and Lang, 2020).
In tomato, DNA methylation is tightly associated with development and fruit ripening (Seymour et al., 2013; Giovannoni et al., 2017; Zuo et al., 2020). Genome-wide mapping of tomato DNA methylome revealed that ripened fruit were governed with low DNA methylation levels (Zhong et al., 2013). Reduced DNA methylation in Slmet1 knockout mutant, encoding a methyltransferase, resulted in defective inflorescence and small leaves (Yang et al., 2019). Moreover, loss-of-function of SlDML2 inhibited fruit ripening by enhancing DNA methylation of ripening-induced genes, indicating the critical role of DNA methylation variation in fruit ripening (Lang et al., 2017). The ascorbic acid is synthesized along with fruit development and ripening. However, whether and how L-ascorbic acid accumulation is regulated by DNA methylation in tomato has not been elucidated. Here, we aimed to explore the link between DNA methylation and AsA accumulation.