Seasonal abscisic acid signal and a basic leucine zipper transcription factor, DkbZIP5, regulate proanthocyanidin biosynthesis in persimmon fruit

Abstract

Proanthocyanidins (PAs) are secondary metabolites that contribute to plant protection and crop quality. Persimmon (Diospyros kaki) has a unique characteristic of accumulating large amounts of PAs, particularly in its fruit. Normal astringent-type and mutant nonastringent-type fruits show different PA accumulation patterns depending on the seasonal expression patterns of DkMyb4, which is a Myb transcription factor (TF) regulating many PA pathway genes in persimmon. In this study, attempts were made to identify the factors involved in DkMyb4 expression and the resultant PA accumulation in persimmon fruit. Treatment with abscisic acid (ABA) and an ABA biosynthesis inhibitor resulted in differential changes in the expression patterns of DkMyb4 and PA biosynthesis in astringent-type and nonastringent-type fruits depending on the development stage. To obtain an ABA-signaling TF, we isolated a full-length basic leucine zipper (bZIP) TF, DkbZIP5, which is highly expressed in persimmon fruit. We also showed that ectopic DkbZIP5 overexpression in persimmon calluses induced the up-regulation of DkMyb4 and the resultant PA biosynthesis. In addition, a detailed molecular characterization using the electrophoretic mobility shift assay and transient reporter assay indicated that DkbZIP5 recognized ABA-responsive elements in the promoter region of DkMyb4 and acted as a direct regulator of DkMyb4 in an ABA-dependent manner. These results suggest that ABA signals may be involved in PA biosynthesis in persimmon fruit via DkMyb4 activation by DkbZIP5.

Figure 1.

Representative scheme of the PA biosynthetic pathway. CHS, Chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′5′-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol 4-reductase; GTs, glycosyltransferases; OMT, O-methyltransferase.

Figure 2.

ABA treatment affects the expression patterns of genes regulating PA biosynthesis and PA concentration in persimmon. A to C, qRT-PCR analysis of gene expression in the control fruit and fruits treated with abamine and S-ABA. A, DkMyb4 expression. B, DkANR expression. C, DkF3′5′H expression. Each treatment is shown as a black point and solid line (control), white point and dotted line (abamine), and gray point and dotted/dashed line (S-ABA) in cv Kuramitsu (A type; squares), Suruga (NA type; circles), and Okugosho (NA type; diamonds). Three fruits of each cultivar were sampled at 1, 3, 5, 7, 9, and 13 WAB and independently subjected to qRT-PCR analysis. The expression levels of each gene are given as values relative to those in cv Kuramitsu at 1 WAB, whose expression level is defined as “100.” Error bars indicate sd (n = 3). D, Concentration of each PA component and total PA of the three cultivars in each treatment at 13 WAB. Main PA components of persimmon fruit, catechin (C), gallocatechin (GC), epicatechin (EC), EGC, epicatechin-gallate (EC-G), and EGC-G (Akagi et al., 2009a), were detected after depolymerization of PA by acid catalysis in the presence of excess phloroglucinol (see “Materials and Methods”). DW, Dry weight. Error bars indicate sd (n = 4). In all panels, asterisks indicate significant differences (*P < 0.05, **P < 0.01) according to Student’s t test compared with the control sections.

Figure 3.

In vitro ABA treatment affects DkMyb4 expression differentially in A- and NA-type cultivars. Relative DkMyb4 expression levels in the control fruit (immediately after sampling) and fruits treated with 0, 0.1, and 10 μm ABA are shown. Each of the three A- and NA-type samples from the Atf lines are displayed: 1 WAB (A) and 9 WAB (B). Fruits from three A-type (Atf-14, Atf-16, and Atf-148) and NA-type (Atf-108, Atf-117, and Atf-145) samples from the Atf lines were independently used for expression analysis. The expression levels are given as values relative to those in Atf-14 in the control at 1 WAB, whose expression level is defined as “100.” Error bars indicate sd (n = 3). Asterisk indicate significant differences (* P < 0.05, ** P < 0.01) according to Student’s t test among the A- and NA-type individuals. [See online article for color version of this figure.]

Figure 4.

The gene tree of bZIP TFs is organized according to ABRE-binding proteins (AREB). AREB-like bZIP TFs in Arabidopsis selected according to previous reports (Jakoby et al., 2002; Suzuki et al., 2003), AREB-like bZIP TFs in other crops studied previously, and DkbZIP5 were used for constructing the tree. At5g24800, which is located on the subclade adjacent to Arabidopsis AREBs (Suzuki et al., 2003), was used as an outgroup gene. The tree is based on the alignment of each full-length bZIP TF using the ClustalW multiple sequence alignment system (Thompson et al., 1997) and the default parameters of the DNA Data Bank of Japan. The tree is constructed using the neighbor-joining method of TreeView (Page, 1996). The scale bar represents 0.5 substitutions per site, and the numbers next to the nodes are bootstrap values from 1,000 replicates. GenBank accession numbers for bZIP TFs in persimmon are provided in “Materials and Methods.”

Figure 5.

qRT-PCR analysis of the expression levels of three AREB-like DkbZIPs isolated from persimmon fruit. A, Temporal expression of DkbZIP2, DkbZIP3, and DkbZIP5 in fruits obtained from 1 WAB until 13 WAB is displayed for each of the three A- and NA-type samples from the Atf lines (Atf-14, Atf-16, and Atf-148 for A type and Atf-108, Atf-117, and Atf-145 for NA type). Mean values of the expression levels in these three individuals are provided for the A- and NA-type cultivars. The expression levels are given as values relative to those in the A type at 1 WAB, whose expression level is defined as “100.” B, Expression levels of DkbZIP2, DkbZIP3, and DkbZIP5 in the stem (9 WAB), young leaf (0 WAB), mature leaf (9 WAB), calyx (9 WAB), fruit flesh (9 WAB), and seed (9 WAB) of the A-type cultivar. Mean values of the expression levels in these three individuals are given for the A- and NA-type cultivars. The expression levels are given as values relative to those in stem, whose expression level is defined as “100.” Error bars indicate sd.

Figure 6.

Functional analysis of DkbZIP5 with persimmon transformation. Some of the persimmon calluses in which sense DkbZIP5 had been introduced were regenerated and designated SbZ5 lines. A, Calluses of SbZ5-1, one of the SbZ5 lines, and control calluses at 6 months after infection of leaves with Agrobacterium (left) and stained with DMACA (right). DMACA staining of the SbZ5-1 calluses tended to have a deep blue color that differed from the control calluses, which indicates a higher PA concentration in the surface cells of the SbZ5-1 calluses. Bars = 2 mm. B, Concentration of each PA component and total PA in the calluses of three SbZ5 and control lines. Error bars indicate sd (n = 4–6). Abbreviations are as in Figure 2D. C, qRT-PCR analysis of gene expression in the calluses of the SbZ5 and control lines. DkbZIP5, DkMyb4, DkMyb2, and two of the structural genes of the PA biosynthetic pathway is given. Expression levels of other structural genes of the PA pathway are shown in Supplemental Figure S4. The expression levels are given as values relative to those in fruit flesh of cv Kuramitsu sampled at 9 WAB, whose expression level is defined as “100.” Using qRT-PCR, we analyzed each of the four individuals for all transgenic lines in three technical replicates. Error bars indicate sd (n = 4 for biological replicates). In all figures, asterisks indicate significant differences (* P < 0.05, ** P < 0.01) according to Student’s t test, in comparison with the control lines. [See online article for color version of this figure.]

Figure 7.

ABRE cis-motifs in the DkMyb4 promoter region in persimmon (cv Kuramitsu) and the ability of the DkbZIP5 fusion protein to bind to them. A, ABRE (ACGTG) motifs in the DkMyb4 promoter region detected using the PLACE database (Higo et al., 1999). * The sequence of ACGCG, another possible ABRE motif, is also given. B, Sequences of oligonucleotides used in EMSA. The ABRE motif from the DkMyb4 promoter region is shown as boldface and underlined nucleotides. The mutated oligonucleotides are shown in red (ACGTG to AGGAT in the ABRE motif). C, EMSA performed using the GST-DkbZIP5 fusion protein and ABRE probe derived from the DkMyb4 promoter region. The binding signals are below the detection level with 150-fold excess nonlabeled oligonucleotide (NLO) of the ABRE motif in comparison with the DIG-labeled oligonucleotide probe; however, the 150-fold excess mutated nonlabeled oligonucleotide (MNLO) of the ABRE motif does not significantly decrease the binding signal.

Figure 8.

DkbZIP5 and the ABA signal activate the DkMyb4 promoter. A, Structures of reporter constructs using the transient assay. pWA-LUC2 and pWNA-LUC contain the whole promoter region (1,874 bp) of DkMyb4 from A-type cv Kuramitsu and NA-type cv Fuyu, respectively, which comprises some single-nucleotide polymorphisms but no differences in their ABRE sequences. Solid red lines in the DkMyb4 promoter regions indicate ABRE cis-motifs. The dotted red line in pR1-LUC2 indicates mutated ABRE, whose changed nucleotides are indicated by boldface. B, Effect of ABA concentration and DkbZIP5 overexpression on the activation of the whole DkMyb4 promoter, using pWA-LUC2 as the reporter. The control indicates analysis using an empty vector as the effector without ABA treatment. C, Effect of the DkMyb4 promoter sequences (presence of each ABRE cis-motif) on activation under DkbZIP5 overexpression and the presence of 100 μm ABA. In all analyses, fold induction that was normalized by LUC2/REN (firefly luciferase activity relative to Renilla luciferase activity) is given. Each column represents the mean value of the three plants containing each of the six technical replicates, for a total of 18 replications, with error bars indicating sd. Asterisks indicate significant differences (* P < 0.05, ** P < 0.01) according to Student’s t test from the control (B) or the noneffector + pWA-LUC2 (C). [See online article for color version of this figure.]