Here, we have isolated a vacuolar iron transporter gene, TgVit1 , from the blue petals of the tulip. The protein TgVit1 is similar in amino acid sequence to AtVIT1 and CCC1, which are known vacuolar iron transporters in plant and yeast, respectively. Analysis of the tissue‐ and stage‐specific expression of TgVit1 and accumulation of TgVit1 revealed a strong correlation between the expression of TgVit1 and blue cell coloration. We characterized the localization of TgVit1 in Welsh onion using a transient expression system, and showed that TgVit1 does indeed localize to vacuoles. We also performed a functional analysis of TgVit1 following transient expression of TgVit1 in purple tulip petal cells, and performed a complementation assay in yeast ccc1 mutants.

We have been studying the mechanism of blue coloration in the tulip, which shows blue coloration at the bottom of the inner perianth and purple coloration in the upper petal ( Figure 1a,b ). We previously reported that both blue and purple cells contain the same anthocyanin, delphinidin 3‐ O‐ rutinoside, as well as a similar composition of co‐pigments, including manghaslin, rutin and mauritianin, and have a similar vacuolar pH of approximately 5.5–5.6 ( Shoji et al. , 2007 ). The only difference between blue and purple cells is their iron content. The concentration of iron ion in blue cells is approximately 10 mm, which is 25 times higher than purple cells ( Shoji et al. , 2007 ). In reconstruction experiments mixed with various vacuolar components, approximately 1 m equivalent of Fe 3+ to delphinidin 3‐ O‐ rutinoside was essential for blue coloration. These findings strongly suggest that a vacuolar iron transporter and/or storage system exists specifically in blue cells of the inner bottom part of the tulip petal and plays an important role in blue flower coloration.

Results and discussion

Isolation of a TgVit1 cDNA from the blue segments of tulip petals The accumulation of iron at the bottom of the inner perianth of the petal of T. gesneriana cv. Murasakizuisho (Shoji et al., 2007) strongly suggests the presence of a vacuolar iron transport and/or storage system in the cells of this region. We sought to isolate the putative tulip vacuolar iron transporter gene from the epidermal cells of the blue segment of the petal. Total RNA was prepared from epidermal cells of the blue segments of tulip petals, and a partial DNA fragment of TgVit1 was amplified by PCR using degenerate primers based on conserved sequences of A. thaliana VIT1 (AtVIT1, At2g01770) and Oryza sativa Vit1 (OsVit1, Os04g0463400). The full‐length cDNA was obtained by 5′ and 3′ RACE. The putative TgVit1 cDNA consisted of 963 bp and encoded a polypeptide of 247 amino acids (Figure 2a). A BLAST search showed that TgVit1 is a member of the CCC1 family; CCC1 is an iron and manganese transporter that transports metals from the cytosol into the vacuole in S. cerevisiae (Li et al., 2001). To date, there have been no reports of a vacuolar iron uptake system in plants other than A. thaliana (AtVIT1; Kim et al., 2006); however, a search of the database revealed several amino acid sequences, including OsVit1/2, PtVit1, VvVit1 and ZmVit1, that were highly homologous to yeast CCC1. Phylogenetic analysis indicated that TgVit1 has a high level of similarity to these putative plant vacuolar iron transporter proteins (Figure 2b). TgVit1 had 85% amino acid similarity to AtVIT1, and 77% identity to OsVit1 (Os04g0463400), its closest homolog to date (Figure 2b). Figure 2 Open in figure viewer PowerPoint The vacuolar iron transporter TgVit1.(a) Alignment of the putative amino acid sequences of vacuolar iron transporters from T. gesneriana (TgVit1) and A. thaliana (AtVIT1). Black and gray boxes indicate identical and similar amino acids, respectively. Putative transmembrane regions are indicated by black lines (I–V). The region used to generate anti‐TgVit1 antibodies is indicated using arrowheads.(b) Phylogenetic relationship of TgVit1 and the vacuolar iron transporter from A. thaliana (AtVIT1, NM_126238), the putative iron transporters from O. sativa (OsVit1, NP001053010 and OsVit2, EAZ09015), Populus trichocarpa (PtVit1, ABK94901), Vitis vinifera (VvVit, CAN83350) and Zea mays, (ZmVit1, DQ245806), and the functional homolog in S. cerevisiae, CCC1 (L24112). The scale bar represents 0.1 substitutions per site. Secondary structure analysis of TgVit1 by the sosui program (Hirokawa et al., 1998) predicted five putative transmembrane domains, all of which exhibited high similarity to those of AtVIT1 (Figure 2a). Thus, the secondary structure of TgVit1 indicates that it is a transmembrane protein, similar to AtVIT1. To determine the number of TgVit genes in tulip, a genomic Southern blot analysis was performed. Genomic DNA was isolated from leaves of the tulip and digested with EcoRI, HindIII or XbaI; then a hybridization experiment was perfomed using a gene‐specific probe. Three to four hybridizing bands were observed in each lane (Figure S1). These data suggest that, in addition to TgVit1, two or three additional TgVit genes may exist in the tulip genome.

Accumulation of TgVit1 protein in blue‐colored cells To further clarify the role of TgVit1 in blue flower coloration, we analyzed the localization of TgVit1 in the tulip plant by immunoblotting. A DNA fragment encoding a TgVit1 polypeptide spanning amino acids 1–27, which was predicted to be the N‐terminal region of the protein (Figure 2a), was expressed in Escherichia coli. Purified recombinant polypeptide was then used to generate rabbit polyclonal antibodies, which were used for immunoblot analysis following affinity purification. We obtained extracts from transgenic Arabidopsis plants that over‐expressed TgVit1 under the control of the CaMV 35S promoter in order to assess the cross‐reactivity of the anti‐TgVit1 polyclonal antibody. Coomassie brilliant blue staining did not reveal any differences in protein levels between transgenic (Figure 4a, lane 2) and wild‐type Arabidopsis (lane 1). When we examined the extracts by immunoblot, the anti‐TgVit1 antibody detected a polypeptide of approximately 32 kDa in transgenic Arabidopsis (lane 4), but not in wild‐type plants (lane 3). These results indicate that the polyclonal anti‐TgVit1 antibody is mono‐specific and can efficiently recognize TgVit1. The calculated molecular weight of TgVit1 (26.2 kDa) is different from the apparent molecular mass detected by immunoblot (32 kDa), which suggested that TgVit1 might be modified in cells. Figure 4 Open in figure viewer PowerPoint Characterization of TgVit1 expression in tulip tissues by immunoblotting.(a) Immunodetection of exogenously expressed TgVit1 in A. thaliana. Soluble protein (10 μg) prepared from cauline leaves was subjected to SDS–PAGE. The gel was stained with Coomassie brilliant blue (left panel, lanes 1 and 2) or analyzed by immunoblotting (right panel, lanes 3 and 4) using an anti‐TgVit1 antibody. Lanes 1 and 3, wild‐type plants; lanes 2 and 4, transgenic plants expressing exogenous TgVit1. Molecular markers are indicated in kDa on the left. The arrowhead indicates TgVit1.(b) Accumulation of TgVit1 in the blue segment of tulip petals. Crude membrane fractions (40 μg of protein) from tulip samples were subjected to SDS–PAGE. Lane 1, parenchyma of purple segments; lane 2, epidermis of purple segments; lane 3, parenchyma of blue segments; lane 4, epidermis of blue segments.(c) Immunoblot analysis of TgVit1 in various tissues of the tulip. Lane 1, leaf; lane 2, stem; lane 3, bulb; lane 4, root.(d) Developmental changes in TgVit1 expression in the epidermis of blue segments of tulip petals. Lane 1, stage 1; lane 2, stage 2; lane 3, stage 3; lane 4, stage 4. We next analyzed crude membrane extracts from various parts of the tulip plant and petal at various flowering stages. In petals, TgVit1 was detected only in blue‐colored epidermal cells from the inner bottom segment (Figure 4b, lane 4), while parenchyma cells of the upper purple‐colored part (lane 1), epidermal cells of the upper purple‐colored part (lane 2), and parenchyma cells of the inner bottom segment (lane 3) were negative for TgVit1. Furthermore, TgVit1 was not detected in leaf (Figure 4c, lane 1), stem (lane 2), bulb (lane 3) or root (lane 4). These results confirm that TgVit1 is expressed only in blue‐colored epidermal cells at the bottom of the inner perianth. Stage‐specific immunoblot analysis of the epidermal cells of the inner bottom segment showed that TgVit1 is highly expressed at stage 1 (Figure 4d, lane 1), and the expression gradually decreased toward stage 4 (lane 4). The expression of TgVit1 protein paralleled the expression of TgVit1 mRNA (Figure 3b). The tissue specificity was also in agreement as expression was detected exclusively in the epidermis of the inner bottom segment. Together, these results strongly suggest that TgVit1 is involved in the blue coloration of cells.

Subcellular localization of TgVit1 We analyzed the subcellular localization of TgVit1 in Welsh onion epidermal cells using an N‐terminal fusion protein of red fluorescent protein (RFP) and TgVit1 (TgVit1::RFP). As a control for vacuolar membrane localization, tonoplast intrinsic protein (γTIP) fused to green fluorescent protein (GFP) (γTIP::GFP) was used (Mitsuhashi et al., 2000). The TgVit1::RFP and γTIP::GFP fusions were introduced into Welsh onion cells, and the subcellular localization was determined by confocal microscopy. In contrast to GFP or RFP alone, TgVit1::RFP was excluded from the nucleus (Figure 5b,c). When TgVit1::RFP and γTIP::GFP were co‐expressed, TgVit1::RFP (RFP fluorescence, Figure 5d) co‐localized with γTIP::GFP (Figure 5e,f). These results indicate that TgVit1 localizes to vacuolar membranes. Figure 5 Open in figure viewer PowerPoint Subcellular localization of TgVit1 in Welsh onion.pTgVit1‐tdTomato and pγTIP‐GFP, as a control, were introduced into Welsh onion epidermal cells simultaneously by particle bombardment. Fluorescence signals were observed using a confocal laser scanning microscope 18 h after bombardment. Scale bar = 20 μm.(a) Bright‐field image.(b) RFP fluorescence from the expression of TgVit1‐tdTomato.(c) Composite image of (a) merged with (b).(d) RFP fluorescence derived from TgVit1‐tdTomato.(e) GFP fluorescence derived from the expression of γTIP‐GFP.(f) Composite image of (d) merged with (e).