Naringenin feeding experiment
Sugars are known to induce anthocyanin pigmentation in Arabidopsis seedlings (Tsukaya et al., 1991). Before the naringenin feeding experiment, the proper concentration of sucrose for the induction of anthocyanin accumulation was determined. In the Col seedlings, as the sucrose concentration increased to 5%, the anthocyanin pigmentation became markedly deeper in the upper hypocotyls and abaxial and marginal regions of the cotyledons. Ten per cent sucrose delayed development, and 20% inhibited germination in Col (data not shown). Two tt19 mutant lines also showed retardation of the germination and seedling development on plates with more than 10% sucrose. Therefore, 5% of sucrose was used in this experiment.
In the Col seedlings, addition of naringenin reinforced anthocyanin pigmentation to some extent (Figure 3a, upper panels). Although some effects were observed in seedling development on the 5% sucrose media, the tt4 mutant exhibited anthocyanin pigmentation as a result of naringenin feeding (Figure 3a, lower panels), as reported previously by Shirley et al. (1995). On the other hand, tt19 did not accumulate anthocyanins despite the naringenin feeding (Figure 3a, middle panels). These results indicate that the TT19 gene functions at a step downstream of the chalcone isomerase (CHI) reaction in the anthocyanin biosynthetic pathway (see Figure 1).
Figure 3. Physiological and histochemical analyses of tt19 mutants.
(a) Photographs of Col, tt19, and tt4 seedlings grown in MS/sucrose/agar plates with or without 0.1 mm naringenin. Scale bar indicates 1 mm.
(b) Depositional patterns of PA precursors in immature seed coat of Col and tt19. Scale bars indicate 50 µm.
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In general, the brown color of the Arabidopsis wild-type testa is thought to be mainly because of the oxidation of PAs (Chapple et al., 1994). To determine the presence and/or distribution of the PA precursors, the tt19 seeds were stained with vanillin (Figure 3b). Vanillin reacts with monomer units of PA precursors and terminal units of PAs in acidic conditions, resulting in the deposition of red pigments in the sites where the PA precursors are accumulated (Deshpande et al., 1986). No reddish color was observed in unstained immature seeds of Col and tt19 (data not shown). At 2–3 days after flowering (DAF), Col and tt19 seeds had clearly different patterns of red pigments: in the Col testa, red pigments appeared to accumulate in large vacuoles in cells of the endothelium layer, while in tt19, they were restricted to a few smaller vacuoles. At 5 DAF, the vacuole of Col was fully expanded within each constitutive cell, whereas such an expanded vacuole was not detected in tt19 (Figure 3b, upper panels). A difference in the distribution pattern of red pigments was continuously detected until 9–10 DAF, after which whole-mount observation was difficult probably because of hardening of the seed coat. To examine the distribution of PA precursors in such a hardened seed coat, we tried to dissect seed coat segments and their most inner (endothelium) layer was observed. Examples at 17 DAF are shown in Figure 3(b, lower panels). In Col, the red pigmentation was very weak and marginal in the constitutive cells of the endothelium layer. In contrast, the red pigmentation was observed throughout each endothelium cell in the tt19 mutants.
Molecular cloning and characterization of the TT19 gene
The TT19 gene was molecularly mapped using F2 individuals derived from crosses of Landsberg erecta with tt19-1 or tt19-2 mutants. Rough mapping suggested that TT19 was localized close to some flavonoid genes such as TT4, TT7, and flavonol synthase 1. However, allelism tests and sequence analyses showed that none of these genes was the TT19 gene (Shikazono et al., 2003). Further mapping was therefore performed using additional F2 individuals. For the tt19-1 allele, DNA markers at 20.6, 23.7, and 25.3 cM of chromosome 5 on the recombinant inbred (RI) map showed a gradual reduction of recombination frequencies, and the TT19 gene was localized at around 29.5 cM. On the other hand, recombination values of the 42.2 and 50.5 cM markers localized the TT19 gene at around 35.5 cM. In addition, recombination was not detected in any of 45 F2 individuals in the region between these two possible locations of the TT19 gene. This phenomenon is often found in mutants induced by ion beams, and suggests that inversion has taken place in this region with the breakpoints around 29.5 and 35.5 cM in the tt19-1 mutant. For the tt19-2 allele, a gradual decline of recombination values into a chromosomal site at around 35 cM was obtained among 103 F2 individuals. These results suggested that the TT19 gene is located in the vicinity of 35 cM, and that in the tt19-1 mutant, one of the breakpoints of the assumed large inversion coincided with the TT19 locus.
We first focused on about five bacterial artificial chromosome (BAC) or P1 clones, to which the map position of the tt19-2 allele has been narrowed down. As the primary characteristic of tt19 mutants is that they have little or no anthocyanins, the TT19 gene appears to be involved in the synthesis and/or accumulation of anthocyanins. In support of this hypothesis, several TT19 candidates could be found from DNA sequences of the five BAC or P1 clones.
Based on the mapping data from the tt19-2 allele, we started to analyze the most probable candidate (identified as a GST-like gene on P1 clone MKP11). The GST-like gene was amplified by PCR using four primer sets covering the whole gene (Figure 4a). Two out of four fragments (f1-r1 and f2-r2) were not amplified in tt19-1, suggesting that these non-amplified DNA regions of the tt19-1 mutant include a breakpoint of the putative large inversion. To isolate the DNA fragment including the rearranged point, thermal asymmetric interlaced (TAIL)-PCR was carried out. The sequence of the TAIL-PCR product demonstrated that the region downstream of the GST-like gene was joined to sequences that are completely homologous to those of BAC F18O22 on chromosome 5 in the reverse direction, with a filler-DNA-like 13-bp sequence at the border. The other rejoining point of the inversion was amplified by TAIL-PCR and sequenced, and was found to contain F18O22 and MKP11 sequences. A 7-bp filler-DNA-like segment is present at the border. Therefore, we concluded that in the tt19-1 mutant, inversion had occurred with the breakpoints in F18O22 and in the second intron of GST-like gene on MKP11 (Figure 4a). The size of this inversion was estimated at about 1000 kbp based on the Arabidopsis genome database. Genomic sequence data indicate that no gene is located at the breakpoint on BAC F18O22.
Figure 4. Isolation and characterization of TT19 gene.
(a) Schematic representation of mutations in tt19-1 and tt19-2. Four primer sets are indicated by the pairs of facing black arrows, which are located directly below their positions in the diagram above.
(b) Molecular complementation of tt19 mutants with wild-type TT19 gene with its authentic promoter. T1 seedling on the sucrose media (upper) and seed color at the ripening stage (lower) are shown. Scale bars indicate 1 mm.
(c) Neighbor-joining tree of the deduced amino acid sequences of TT19, Arabidopsis EST H36860, Arabidopsis GST6, Arabidopsis ERD13, petunia AN9, maize BZ2, and parsley GST1. Maize BZ2 and parsley GST1 were used as outgroups. Bootstrap values are indicated at each branch.
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PCR analysis was also carried out for the GST-like locus in the tt19-2 mutant. The f1 and r1 primers did not amplify a product, suggesting that the tt19-2 mutant underwent DNA rearrangement with a breakpoint in the f1-r1 region. TAIL-PCR revealed that the nucleotide at position −53 (based on defining the translation initiation site as +1) was rejoined to a region 16.7 kbp further upstream (Figure 4a). No other mutation was found on the GST-like gene from the rejoined site to 60 bp downstream of exon 3. As in the case of the tt19-1 mutant, the genomic sequence data indicate that there is no gene on the upper breakpoint of 16.7-kbp fragment of the tt19-2 allele.
As both tt19 mutants have mutations in the GST-like gene, molecular complementation of the tt19 mutants was carried out using the wild-type GST-like gene with its authentic promoter (about 2.4 kbp in length). In each of the five independent T1 plants (named the tt19/TT19 line), anthocyanin in the seedlings and brown pigmentation in the testa at the ripening stage were both restored to wild-type level (Figure 4b). This is conclusive evidence that disruption of the GST-like gene is responsible for the flavonoid-deficient phenotype of the tt19 mutants. In the following, this gene is referred to as TT19. RT-PCR revealed that expression of the TT19 gene was abolished in the two tt19 mutants (data not shown).
Plant GSTs are divided on the basis of sequence identity into five types (Phi, Tau, Theta, Zeta, and Lambda; Dixon et al., 2002). As the TT19 gene is classified as a type Phi GST gene, the deduced amino acid sequence of wild-type TT19 gene was compared with that of other Arabidopsis Phi GSTs, such as an auxin-inducible GST (GST6; Chen et al., 1996), a dehydration-inducible GST (early-responsive to dehydration (ERD)13; Kiyosue et al., 1993), and an expressed sequence tags (EST) clone H36860 protein (Alfenito et al., 1998). Some flavonoid-related GSTs from other species were also included in the analysis. Petunia AN9 and maize BZ2 are involved in anthocyanin accumulation (Alfenito et al., 1998; Marrs et al., 1995). Parsley GST (PcGST1) is a putative regulator that stimulates chalcone synthase (CHS) expression in the presence of UV light (Loyall et al., 2000). The Arabidopsis H36860 protein is a functional GST with a high amino acid identity to AN9, and was previously found not to complement bz2 (Alfenito et al., 1998). A phylogenetic analysis indicated that TT19 primarily clustered with H36860, and secondarily clustered with AN9, with high bootstrap values (Figure 4c). BZ2 and PcGST1 formed a distinct cluster, which is consistent with their previous classification as type Tau GSTs (Loyall et al., 2000).
tt19 ban double mutant analysis
BANYULS (BAN) encodes anthocyanidin reductase (ANR), one of the enzymes involved specifically in the PA biosynthetic pathway in Arabidopsis (Xie et al., 2003; Figure 1). Loss of function of the BAN gene results in precocious accumulation of anthocyanins and no accumulation of flavan-3-ols in the seed coat (Albert et al., 1997; Devic et al., 1999), which is caused by the change of the flow of competitive substrate (anthocyanidins) from PA synthesis to anthocyanin synthesis (see Figure 1). To analyze the function of TT19 in the flavonoid pathway, a double mutant with tt19 and ban was constructed and its phenotype was characterized.
Pigmentation of anthocyanins was not visible in leaves and stems of the tt19 ban double mutant. The immature seed coat of the double mutant had a very weak coloration but no conspicuous accumulation of anthocyanins (Figure 6a). A vanillin assay did not detect PA precursors in the immature seed coat of the double mutant, as was the case in the ban mutant (Devic et al., 1999) and the ban-4 mutant used here (unpublished data). The seed color of the double mutant at the ripening stage (Figure 6b, third from left) was about the same shade of brown as that of tt19 (second from left), although it was a little grayer than tt19. The gray coloration of the double mutant seems to be because of the leakage of some of the anthocyanins during the immature stage (Figure 6a). One of the characteristics of tt19 is a darkening of seed color during long-term desiccation. In contrast, no change in seed color was observed in the double mutant after an additional desiccation period (Figure 6c).
Figure 6. Testa phenotype of tt19 ban double mutants.
In each panel, order from left to right is Col, tt19, tt19 ban double mutant, and ban. Scale bars indicate 1 mm.
(a) 8 DAF immature siliques.
(b) 20 DAF mature siliques.
(c) Seed color after an additional 7-week desiccation after the ripening stage.
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