MdGSTF6, activated by MdMYB1, plays an essential role in anthocyanin accumulation in apple

Abstract

Anthocyanins are biosynthesized on the cytosolic surface of the endoplasmic reticulum and then transported into the vacuole for storage. Glutathione S-transferases (GSTs) are considered to be responsible for the transport of anthocyanins into the vacuole. However, the regulatory mechanisms of GSTs in plants are still unclear. Here, we performed a genome-wide analysis and identified 69 GST genes in apple. The expression of MdGSTF6 was positively correlated with the anthocyanin content (r = 0.949) during 'Yanfu 8' fruit development. The overexpression of MdGSTF6 in the Arabidopsis thaliana tt19 mutant resulted in seedlings of 35S::MdGSTF6-GFP/tt19 that could accumulate anthocyanin and rescue its phenotype, suggesting that MdGSTF6 was an anthocyanin transporter. The silencing of MdGSTF6 affected anthocyanin accumulation in apple fruit. Moreover, the knockdown of MdGSTF6 by RNA interference in cultured 'Gala' seedlings inhibited anthocyanin accumulation. The interaction experiments showed that MdMYB1 could bind directly to the MdGSTF6 promoter to transcriptionally activate its expression. Collectively, our results demonstrate that MdGSTF6 encodes an important GST transporter of anthocyanins in apple fruit and provide evidence for the associated regulatory mechanisms. Therefore, MdMYB1 can not only regulate anthocyanin synthesis, but also control the transport of anthocyanin in apples. This information may be useful for further clarifying the regulation of anthocyanin transport in apple.

Fig. 1. Phylogenetic tree of glutathione S-transferase (GST) proteins in apple, Arabidopsis thaliana, maize, and dragon’s blood tree (Dracaena draco).

The 69 apple, 63 A. thaliana, 29 maize, and 20 D. draco GST protein sequences were aligned using Clustal W. The phylogenetic tree was constructed based on the neighbor-joining algorithm. GST proteins were clustered into nine classes. The protein sequences are listed in Table S3

Fig. 2. Fruit development and anthocyanin accumulation in ‘Yanfu 8’ apple cultivars.

a Fruit development of 'Yanfu 8’ at the indicated time points. b Anthocyanin contents in fruit peels of ‘Yanfu 8’. c Cyanidin-3-galactoside contents in fruit peels of ‘Yanfu 8’. Scale bar: 1 cm. d Expression of 4 glutathione S-transferase (GST) genes during anthocyanin accumulation in apple. Expression levels were calculated relative to apple actin. Three biological replicates of each sample were analyzed. Data are expressed as the means ± SD, n = 3. The different letters denote significant differences according to one-way analysis of variance (ANOVA) (P < 0.05)

Fig. 3. Phylogenetic analysis and subcellular localization of MdGSTF6.

a Phylogenetic tree analysis of glutathione S-transferases (GSTs). The protein sequences are listed in Table S3. b Sequence alignment of seven GSTs. TT19 in Arabidopsis thaliana (NC_003076.8), Riant in peach (ALE31200.1), RAP in strawberry, GSTF12 in grape (NP_001267869.1), AN9 in petunia (CAA68993.1), and BZ2 in maize (NP_001183661.1) were used to build the phylogenetic tree and for sequence alignment analysis. c Subcellular localization of the 35S::MdGSTF6-GFP fusion construct in apple callus protoplasts. Protoplasts expressing 35S::GFP were used as a control

Fig. 4. Phenotypes of 35S::MdGSTF6-GFP transgenic lines in Arabidopsis thaliana tt19.

a Phenotypes of 7-day-old seedlings grown on Murashige and Skoog (MS) medium with 6% sucrose. Scale bar: 4 mm. b Images of A. thaliana seeds. Images of wild-type (WT), tt19, and three transgenic lines of 35S::MdGSTF6-GFP (lines 2, 5, and 7) in a tt19 background are presented. Scale bar: 1 mm

Fig. 5. MdGSTF6 silencing inhibits anthocyanin accumulation in apple peels.

a Phenotypes of ‘Fuji’ fruits after silencing MdGSTF6. Empty pTRV2 was used as a control. b Abundance of MdGSTF6 protein in MdGSTF6-silenced apple and control c Transcript levels of genes in anthocyanin pathway in MdGSTF6-silenced apple and control. CHS chalcone synthase, CHI chalcone isomerase, F3H flavanone 3-hydroxylase, DFR dihydroflavonol 4-reductase, ANS anthocyanidin synthase, UFGT UDP-glucose: flavonoid-3-O-glucosyltransferase, GST glutathione S-transferase MdGSTF6. Data are expressed as the means ± SD, n = 3. The asterisks denote significant differences according to one-way analysis of variance (ANOVA) (**P < 0.01)

Fig. 6. MdGSTF6 silencing inhibits anthocyanin accumulation in ‘Royal Gala’ transgenic lines.

a Phenotype of wild-type (WT) and three MdGSTF6-RNAi lines (#5, #6, and #8) in response to sucrose treatment (6%). b Leaves of WT and three transgenic ‘Royal Gala’ lines under sucrose treatment. c Abundance of MdGSTF6 protein in WT and three transgenic ‘Royal Gala’ lines. Plant total proteins were visualized with Coomassie brilliant blue staining. d Anthocyanin contents in WT and three transgenic ‘Royal Gala’ lines. Data are expressed as the means ± SD, n = 3. The different letters denote significant differences according to one-way analysis of variance (ANOVA) (P < 0.05)

Fig. 7. MdMYB1 bound to the MdGSTF6 promoter.

a Y1H assay showing the interaction between MdMYB1 and the MdGSTF6 promoter. The empty pGADT7 vector was used as a control. b Chromatin immunoprecipitation (ChIP) assay showing binding of MdMYB1 to the G1 region of the MdGSTF6 promoter in vivo. Apple ‘Orin’ calli overexpressing HA protein was used as a control. c Electrophoretic mobility shift assay (EMSA) result showing the interaction between MdMYB1 and labeled probes in the MdGSTF6 promoter. Red letters indicate MYB-binding site (MBS) and mutated MBS (MdGSTF6m). Lane 2 contains labeled DNA probes and MdMYB1 protein without a competitor. Increasing amounts (10×, 25×, and 50×) of unlabeled normal DNA probes (MdGSTF6 in lanes 3, 4, and 5 of each blot) or unlabeled mutated DNA probes (MdGSTF6m in lanes 7, 8, and 9 of each blot) were added as cold competitors. Lane 6 contains labeled mutated DNA probes and the MdMYB1 protein

Fig. 8. MdMYB1 activates transcription of MdGSTF6 to enhance its expression.

a Effects of MdMYB1, MdbHLH3, and MdbHLH33 individually and in combination on the promoter activity of MdGSTF6 in a luciferase reporter assay. b β-Glucuronidase (GUS) staining of pMdGSTF6::GUS and pMdGSTF6::GUS plus 35S::MdMYB1-HA. c GUS activity in apple calli as labeled. GUS activity was measured three times with three replicates

Fig. 9

Model for the transcriptional regulation of anthocyanin biosynthesis and transport in apple. DFR dihydroflavonol 4-reductase, ANS leucoanthocyanidin dioxygenase, UFGT UDP-glucose: flavonoid-3-O-glucosyltransferase, GST glutathione S-transferase