Expression of <i>HmVALT</i> and <i>HmPALT</i>1 and accumulation of their gene products in hydrangea.

<p>(<b>A</b>) The amount of <i>HmVALT</i> and <i>HmPALT1</i> mRNA was determined by quantitative RT-PCR. mRNAs were prepared from sepal at each stage and other tissues. The data are relative to the expression of 18S ribosomal RNA and were further normalized to the level of sepal at stage 1 mRNA, which was expressed as 1.0. The error bars represent SD (n = 3). (<b>B</b>) Immunoblot analyses of HmVALT and HmPALT1 in the sepals. Hydrophobic protein fractions were extracted from sepal tissues at each stage and subjected to SDS–PAGE (20 µg of protein).</p

Procedure for the identification of Al transporters in hydrangea sepals.

<p>(<b>A</b>) Blue color development of <i>H. macrophylla</i> sepal. The sepals become blue during maturation, and the color was developed by an Al³⁺ complex of delphinidin 3-glucoside (<b>1</b>) and quinic acid esters (<b>3</b>, <b>4</b>) in the vacuoles of the second layer. The maturation stages were separated into three categories by its coloration as stage 1: colorless, stage 2: starting coloration and stage 3: fully colored. Scale bar of the transverse section = 50 µm. (<b>B</b>) Strategy for the identification of genes encoding a vacuolar Al transporter. The candidate genes were selected and refined by a cDNA microarray analyses and the results of database searches for their functions and subcellular localizations. The screening was conducted using <i>Δhsp150</i> yeast, which had weak cell walls and Al easily invaded into the cell. If the candidate gene encoded a vacuolar Al transport activity, this transformant in <i>Δhsp150</i> yeast should become tolerant of Al by segregating Al into vacuoles.</p

Determination of amino acids responsible for Al transport activity of HmVALT1 or HmPALT1.

<p>(<b>A</b>) Al tolerance assay of amino acid replaced HmVALT transformant. <i>Δhsp150</i> yeast cells that harbored HmVALT-L162T and −G196A were spotted onto LPP medium (pH 3.5) with or without 2 mM Al₂(SO₄)₃, and the plates were incubated at 30°C for 4 d.HmVALT-L162T transformant grew less indicating to become more Al-sensitive but that of HmVALT-G196A grew at similar level. (<b>B</b>) Al tolerance assay of amino acid replaced HmPALT transformant. Wild type yeast cells that harbored HmPALT1-T114G, −E181Δ and −H188P were spotted onto LPP medium (pH 3.5) with or without 1 mM Al₂(SO₄)₃, and the plates were incubated at 30°C for 4 d. HmPALT1-E181Δ transformant was less sensitive to Al, but others showed to be more sensitive.</p

Comparison of HmPALT1 and the NIP proteins.

<p>(<b>A</b>) Phylogenetic tree was generated from a ClustalW alignment of selected NIP and HmPALT1 amino acid sequences using TreeView 1.6.6. (<b>B</b>) Alignment of HmPALT1 and similar amino acid sequences in Figure5A. Black and gray boxes indicate identical and similar amino acids, respectively. The red bars above the alignment denote the positions of the NPA motif. The aligned ar/R selectivity filter residues are highlighted in vertical blue boxes. The positions that are indicated by black arrows were mutated in the analysis of the yeast Al-sensitivity test (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043189#pone-0043189-g008" target="_blank">Figure 8</a>). Sequences acquired from accessions: PtNIP [XP_002297797], RcNIP1;1 [XP_002527308], MtNIP [AAS48063], AtNIP5;1 [NP_192776], HvLsi6 [BAH84977], HvLsi1 [BAH24163].</p

The organic components of <i>H. macrophylla</i> sepals responsible for the blue color development.

<p>All of the colored sepals contained the same anthocyanin component (delphinidin 3-glucoside, <b>1</b>) and co-pigments (chlorogenic acid, <b>2</b>; neochlorogenic acid, <b>3</b>; 5-<i>O-p</i>-coumaroylquinic acid, <b>4</b>). The sepal color is affected by the ratio of co-pigmnets, amount of Al³⁺ and vacuolar pH.</p

Identification of a plasma membrane-localized Al-transporter, HmPALT1, in hydrangea sepals.

<p>(<b>A</b>) Al tolerance assay of HmPALT1. <i>Δhsp150</i> and wild-type (WT) yeast cells carrying <i>HmPALT1</i> or the empty vector (pYES2) were spotted onto LPP–uracil medium (pH 3.5) with or without 1 and 2 mM Al₂(SO₄)₃, and the plates were incubated at 30°C for 4 d. (<b>B</b>) The intracellular Al content of yeast cells that were transformed with <i>HmPALT1</i>. Yeast cells carrying <i>HmPALT1</i> or the empty vector were exposed to different concentrations of Al₂(SO₄)₃ at pH 3.5 for 2 d. The intracellular Al content in both transformants increased with the Al in the medium in a dose-dependent manner. It is noteworthy, however, that the Al content of the yeast carrying <i>HmPALT1</i> was approximately twice as much as that of the vector control cells, which was significant (**<i>P</i><0.01 and *<i>P</i><0.05 by Student's <i>t</i> test, respectively). The error bars represent SE (n = 3). (<b>C</b>) Subcellular localization of HmPALT1. The construct <i>pHmPALT1-mCherry</i> was simultaneously introduced into Welsh onion epidermal cells by particle bombardment with <i>pPIP1A-GFP</i> as a plasma membrane marker. The fluorescent signals were observed under microscopy 21 h after the bombardment. The merged image shows that HmPALT1 localizes to the plasma membrane. Scale bar = 50 µm.</p

Effect of overexpression of <i>HmVALT</i> and/or <i>HmPALT1</i> in Arabidopsis.

<p>(<b>A</b>) The three independent lines of <i>HmVALT</i>- and/or <i>HmPALT1</i>-overexpressing Arabidopsis were grown in Al medium with 0 or 0.75 mM AlCl₃ for 5 d. The mRNA expression of each gene was confirmed by RT-PCR. <i>HmVALT</i>, <i>HmPALT1</i> and <i>HmVALT</i> and <i>HmPALT1</i>-overexpressing plants were designated as OXVALT, OXPALT1, OXVALT+PALT1, respectively. (B) The root length of <i>HmVALT</i>- and/or <i>HmPALT1</i>-overexpressing Arabidopsis was measured with 0 or 0.75 mM AlCl₃ for 5 d. The ratio of root length in plants treated with 0.75 mM AlCl₃ to root length in plants without aluminum was displayed as the relative root length in the figure. Statistical significances were observed between the transgenic line and the non-transgenic line by Student's <i>t</i> test (**<i>P</i><0.01 and *<i>P</i><0.05). Error bars represent SE (each line was examined by n = 6). (C) The change of relative root length treated with various Al concentrations (0, 0.5, 0.75 and 1 mM). The each relative root length was shown as the average among three idependent lines of the same transgene. Error bars represent SE (n = 18).</p