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Background The health benefits of anthocyanins impel researchers and food producers to explorer new methods to increase anthocyanin contents in plant foods. Our previous studies revealed a positive role of nitric oxide (NO) in anthocyanin accumulation in radish (Raphanus sativus L.) sprouts. The application of hemin, an inducer of heme oxygenase-1 (HO-1), can effectively elevate NO production in vivo. Hemin treatment also improves plant growth and stress tolerance. This study is aimed to assess the effects of hemin treatment on anthocyanin production in radish sprouts, and to investigate whether NO signalling is involved in this process. Results The application of hemin significantly up regulated the expressions of many anthocyanins biosynthesis related structure and regulatory genes, leading to increased anthocyanins accumulation in radish hypocotyls. Hemin treatment also raised NO contents in radish sprouts, probably through enhancing nitrate reductase (NR) activity and Nitric Oxide-Associated 1 (NOA1) expression. Comparing the effects of Zinc Protoporphyrin (ZnPP, HO-1 activity inhibitor), Sodium Nitroprusside (SNP, NO donor) and carboxy-PTIO (cPTIO, NO-scavenger) on anthocyanin and NO production, a positive role of NO signalling has been revealed in hemin-derived anthocyanin accumulation. A positive feedback loop between HO-1 and NO may be involved in regulating this process. Conclusions Hemin induced anthocyanin accumulation in radish sprouts through HO-1 and NO signalling network.

Anthocyanins are water-soluble plant pigments mainly stored in the plant vacuoles. Glutathione S-transferases (GSTs) are a multifunctional enzyme family, which can regulate substance metabolism and biological and abiotic stresses in plants. However, few reports were focused on the involvement of GSTs in anthocyanin sequestration in red skin radish. Here, we identified a glutathione S-transferase gene RsGSTF12 that played roles in anthocyanin sequestration in radish. The bioinformatics analysis revealed that RsGSTF12 belonged to the phi (F) class of glutathione S-transferases and showed a high homology with AtGSTF12, followed by AtGSTF11. The subcellular localization assay showed that RsGSTF12 was located in the endoplasmic reticulum and tonoplast. Temporal and spatial gene expression-specific analyses uncovered a strong correlation of RsGSTF12 with anthocyanin accumulation in radish sprouts. The anthocyanin solubility assay found RsGSTF12 was capable of improving cyanidin water solubility in vitro. Transiently expressing RsGSTF12 in radish cotyledons was able to increase their anthocyanin sequestrations. Furthermore, the functional complementation and overexpression of the Arabidopsis thaliana tt19 mutant and wild type demonstrated that RsGSTF12 might play an indispensable role in anthocyanin accumulation in radish. Taken together, we provide compelling evidence that RsGSTF12 functions critically in how anthocyanins are sequestrated in radish, which may enrich our understanding of the mechanism of anthocyanin sequestration.

Background: The biosynthesis of anthocyanin in the hypocotyls of radish (Raphanus sativus L.) sprouts was enhanced by hemin in our preliminary experiments, but the underlying mechanism is unclear. Here, we found that NO (nitric oxide) exerted an essential role in Hemin-regulated anthocyanin biosynthesis, which was supported by the following results.Results: Hemin boosted anthocyanin as well as NO content. NO-scavenger cPTIO (carboxy-PTIO) significantly attenuated hemin-induced increase of anthocyanin content, transcripts of anthocyanin synthesis related genes and positive transcription factors, implying that NO played a prominent role during hemin-induced anthocyanin biosynthesis. Hemin specific inhibitor ZnPP (Zinc Protoporphyrin) strongly reduced anthocyanin content, while, NO donor SNP (Sodium Nitroprusside) addition considerably reversed this inhibition and by contrast, resulted in a significant increase in anthocyanin accumulation, closely paralleling the transcripts of structural genes and transcription factors. Moreover, NO content, NR (nitrate reductase) activity and expression level of NOA (nitric oxide associated factor) were up-regulated by Hemin. Conclusions:Those consequences indicated that NO might work downstream in Hemin-heightened anthocyanin accumulation in radish sprouts.

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