DNA demethylation is involved in nitric oxide-induced flowering in tomato

Flowering is one of the most important phenological periods, as it determines the timing of fruit maturation and seed dispersal. To date, both nitric oxide (NO) and DNA demethylation have been reported to regulate flowering in plants. However, there is no compelling experimental evidence for a relationship between NO and DNA demethylation during plant flowering. In this study, an NO donor and a DNA methylation inhibitor were used to investigate the involvement of DNA demethylation in NO-mediated tomato (Solanum lycopersicum cv. Micro-Tom) flowering. The results showed that the promoting effect of NO on tomato flowering was dose-dependent, with the greatest positive effect observed at 10 μmol L^–1 of the NO donor S-nitrosoglutathione (GSNO). Treatment with 50 μmol L^–1 of the DNA methylation inhibitor 5-azacitidine (5-AzaC) also significantly promoted tomato flowering. Moreover, GSNO and 5-AzaC increased the peroxidase (POD) and catalase (CAT) activities and cytokinin (CTK) and proline contents, while they reduced the gibberellic acid (GA3) and indole-3-acetic acid (IAA) contents. Co-treatment with GSNO and 5-AzaC accelerated the positive effects of GSNO and 5-AzaC in promoting tomato flowering. Meanwhile, compared with a GSNO or 5-AzaC treatment alone, co-treatment with GSNO+5-AzaC significantly increased the global DNA demethylation levels in different tissues of tomato. The results also indicate that DNA demethylation may be involved in NO-induced flowering. The expression of flowering genes was significantly altered by the GSNO+5-AzaC treatment. Five of these flowering induction genes, ARGONAUTE 4 (AGO4A), SlSP3D/SINGLE FLOWER TRUSS (SFT), MutS HOMOLOG 1 (MSH1), ZINC FINGER PROTEIN 2 (ZFP2), and FLOWERING LOCUS D (FLD), were selected as candidate genes for further study. An McrBC-PCR analysis showed that DNA demethylation of the SFT gene in the apex and the FLD gene in the stem might be involved in NO-induced flowering. Therefore, this study shows that NO might promote tomato flowering by mediating the DNA demethylation of flowering induction genes, and it provides direct evidence for a synergistic effect of NO and DNA demethylation in promoting tomato flowering.