DkMyb4 is a Myb transcription factor involved in proanthocyanidin biosynthesis in persimmon fruit

Abstract

Proanthocyanidins (PAs) are secondary metabolites that contribute to the protection of the plant and also to the taste of the fruit, mainly through astringency. Persimmon (Diospyros kaki) is unique in being able to accumulate abundant PAs in the fruit flesh. Fruits of the nonastringent (NA)-type mutants lose their ability to produce PA at an early stage of fruit development, while those of the normal astringent (A) type remain rich in PA until fully ripened. The expression of many PA pathway genes was coincidentally terminated in the NA type at an early stage of fruit development. The five genes encoding the Myb transcription factor were isolated from an A-type cultivar (Kuramitsu). One of them, DkMyb4, showed an expression pattern synchronous to that of the PA pathway genes in A- and NA-type fruit flesh. The ectopic expression of DkMyb4 in kiwifruit (Actinidia deliciosa) induced PA biosynthesis but not anthocyanin biosynthesis. The suppression of DkMyb4 in persimmon calluses caused a substantial down-regulation of the PA pathway genes and PA biosynthesis. Furthermore, analysis of the DNA-binding ability of DkMyb4 showed that it directly binds to the MYBCORE cis-motif in the promoters of the some PA pathway genes. All our results indicate that DkMyb4 acts as a regulator of PA biosynthesis in persimmon and, therefore, suggest that the reduction in the DkMyb4 expression causes the NA-type-specific down-regulation of PA biosynthesis and resultant NA trait.

Figure 1.

Scheme of the PA biosynthetic pathway. ANR, Anthocyanidin reductase; ANS, anthocyanidin synthase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; DHD/SDH, 3-dehydroquinate dehydratase/shikimate 5-dehydrogenase; F3H, flavanone 3-hydroxylase; F3′H, flavanone 3′-hydroxylase; F3′5′H, flavanone 3′5′-hydroxylase; FLS, flavonol synthase; GTs, glycosyltransferases; LAR, leucoanthocyanidin reductase; OMT, O-methyltransferase; PAL, Phe ammonia lyase.

Figure 2.

Development patterns of fruits in A and NA individuals of the Atf line. Circles and dotted lines indicate NA-type individuals, and squares and solid lines indicate A-type individuals. A, Total PA concentration per fresh weight. B, Total PA content per fruit. Data collection started at 3 WAB and continued until 11 or 13 WAB. Error bars indicate sd. We analyzed three independent fruits for each individual of the Atf line (n = 3).

Figure 3.

qRT-PCR analysis for determining the expression levels of the structural genes involved in PA biosynthesis. Flesh from four A-type (Atf-31, Atf-45, Atf-47, and Atf-148) and NA-type (Atf-9, Atf-36, Atf-117, and Atf-145) individual fruits from 3 to 13 WAB was used for the expression analysis. The expression level is shown as the value relative to expression of DkActin (accession no. AB473616) in each sample. Expression is shown for genes encoding the enzymes PAL, CHS, CHI, F3H, F3′H, F3′5′H, DFR, ANS, LAR, ANR, and DHD/SDH. Error bars indicate sd.

Figure 4.

Comparison of DkMyb1 to -4 and DksMyb1 deduced amino acid sequences with other Myb TFs. A, Protein sequence alignment of the global Myb TFs by phylogenetic analysis. The R2 and R3 domains are shown, and the [D/E]Lx₂[R/K]x₃Lx₆Lx₃R motif interacting with R-like bHLH is indicated in the box. B, Phylogenetic analysis displaying the similarities of DkMyb1 to -4 with other Myb TFs. The tree is based on the alignment of the 105 amino acids spanning the R2R3 domain with the ClustalW multiple sequence alignment system (Thompson et al., 1997) using the default parameters of the DNA Data Bank of Japan. The tree was constructed using the neighbor-joining method of TreeView (Page, 1996). The scale bar represents 0.1 substitutions per site, and the numbers next to the nodes are bootstrap values from 1,000 replicates. The GenBank accession numbers of Myb TFs are as follows: AtMYB111 (NP_199744), AtMYB12 (NP_182268), ZmMYB31 (AM156906), ZmMYB42 (AM156908), DkMYB1 (AB503698), DkMYB4 (AB503671), VvMybPA1 (CAJ90831), PmMBF1 (AAA82943), DkMYB2 (AB503699), VvMybPA2 (EU919682), AtMYB123-TT2 (Q9FJA2), AtMYB15 (Y14207), DkMYB3 (AB503670), ODO1 (AAV98200), AtMyb4 (BAA21619), MdMYB8 (DQ267899), AtMYB23-GL1 (NP_189430), AtMYB66-WER (AAF18939), AmROSEA2 (ABB83827), AmROSEA1 (ABB83826), AmVENOSA (ABB83828), CaE (AJ608992), LeANT1 (AAQ55181), PhAN2 (AAF66727), AtMYB90-PAP2 (NP_176813), AtMYB75-PAP1 (NP_176057), VvMYBA1 (BAD18977), VvMYBA2 (BAD18978), and MdMYB10 (ABB84753). [See online article for color version of this figure.]

Figure 5.

qRT-PCR analysis of expression levels of the Myb TFs isolated from persimmon fruit. A, Temporal expression for DkMyb1 to -4 and DksMyb1 from 3 to 13 WAB in four individuals of A or NA type. B, Gene expression of DkMyb4 in the fruit flesh of eight A-type cultivars and six NA-type cultivars at 7 WAB. C, Gene expression of DkMyb4 in fruit flesh, leaf, stem, fruit calyx, and fruit seed of two A- and NA-type cultivars at 7 WAB. Error bars indicate sd. [See online article for color version of this figure.]

Figure 6.

Functional analysis of DkMyb4 with kiwifruit transformation. Ectopic expression of DkMyb4-induced PA accumulation in the kiwifruit callus. Growth of the DkMyb4-introduced callus is shown after the in vitro infection of leaves with Agrobacterium. A, Four weeks. B, Six weeks. C, Eight weeks. D, Ten weeks. E and F, Three months. G and H, Control callus with empty vector after 3 months. I, DkMyb4-introduced and control calluses at 3 months after infection are stained with DMACA. The strong blue coloration in the DkMyb4-introduced callus indicates the accumulation of a large amount of PA in the surface cells. J, DkMyb4-introduced and control plants at 6 months after infection. The control callus regenerated, while the DkMyb4-introduced callus is brown and growth has stopped. Bars = 2 mm.

Figure 7.

Functional analysis of DkMyb4 with persimmon transformation. Some of the persimmon calluses into which antisense DkMyb4 had been introduced are regenerated and designated the AM4 lines. A, The AM4 lines and control callus at 2 months after the in vitro infection of leaves with Agrobacterium (left) and stained with DMACA (right). Calluses of the AM4 lines clearly showed less blue staining than controls. This indicates lower PA accumulation in the surface cells of the AM4 line calluses. Bars = 10 mm. B, Soluble PA content (left) and soluble phenol compound content (right) in the AM4 line calluses and control callus. FW, Fresh weight. C, Sense DkMyb4-introduced persimmon callus. All sense DkMyb4 lines have stopped growing and turned black. Bar = 2 mm. D, Gene expression in the AM4 line and control calluses. The structural genes of the PA biosynthetic pathway and DkDHD/SDH are measured. The transcription levels of the genes showing the NA-type-specific down-regulation (Fig. 3) were lower in the AM4 lines than those in the controls. Expression of DkLAR was not detected (N.D). E, PA subunit composition in the AM4 line and control (Cont.) callus. The contents of EC and EGC were significantly lower in the AM4 lines than those in the controls. The contents of catechin (CA) and gallocatechin (GC) were not changed in the AM4 lines. Error bars indicate sd. We analyzed each individual with three replications by HPLC (n = 3). DW, Dry weight. * The galloylated EC and EGC were also detected slightly in both AM4 lines and controls and contained in EC and EGC, respectively, in E.

Figure 8.

MYB-binding cis-motifs in the promoter region of the PA pathway genes in persimmon (Kuramitsu) and the ability of the DkMyb4 fusion protein to bind to them. A, MYB-binding motifs in the promoters of DkF3′H, DkF3′5′H, DkANS, DkLAR, and DkANR detected in the sense coding strand using the PLACE database (http://www.dna.affrc.go.jp/PLACE/signalup.html; Higo et al., 1999). B, Sequences for oligonucleotides used in EMSA analysis. The MYB-binding motifs are shown as the underlined nucleotides. The mutated oligonucleotide sequences of the MYBCORE cis-motif are shown in boldface (G and T to C and A). C, EMSA using DkMyb4 fusion protein and the MYBCORE cis-motif derived from the promoter region of DkF3′5′H (top). The binding signals are reduced in proportion to the amount of nonlabeled MYBCORE inhibitor at 20-, 50-, or 150-fold excess versus the DIG-labeled oligonucleotide probe, but 150-fold excess nonlabeled mutated MYBCORE inhibitor does not significantly decrease the binding signal. The binding signals were detected also in EMSA using DkMyb4 fusion protein and the MYBCORE cis-motif derived from the promoter region of DkANR and DkANS (bottom). D, EMSA using DkMyb4 fusion protein and the MYB1AT, MYB2CONSENSUS, MYBPLANT, and MYB1ST motifs. Significant binding signals are not detected in any of these.

Figure 9.

MYB-binding cis-motifs in the promoter region of ANR orthologs from persimmon (cv Kuramitsu), grapevine (cv Pinot Noir), and Arabidopsis. Analysis was performed using the PLACE database as described in the legend to Figure 8.