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First published online October 24, 2008; 10.1105/tpc.108.058628 The Plant Cell 20:2860-2875 (2008) © 2008 American Society of Plant Biologists NIP6;1 Is a Boric Acid Channel for Preferential Transport of Boron to Growing Shoot Tissues in Arabidopsis[W]
a Graduate School of Agricultural and Life Science, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 1 Address correspondence to atorufu{at}mail.ecc.u-tokyo.ac.jp.
Boron (B) in soil is taken up by roots through NIP5;1, a boric acid channel, and is loaded into the xylem by BOR1, a borate exporter. Here, the function of Arabidopsis thaliana NIP6;1, the most similar gene to NIP5;1, was studied. NIP6;1 facilitates the rapid permeation of boric acid across the membrane but is completely impermeable to water. NIP6;1 transcript accumulation is elevated in response to B deprivation in shoots but not in roots. NIP6;1 promoter–β-glucuronidase is predominantly expressed in nodal regions of shoots, especially the phloem region of vascular tissues. Three independently identified T-DNA insertion lines for the NIP6;1 gene exhibited reduced expansion of young rosette leaves only under low-B conditions. B concentrations are reduced in young rosette leaves but not in the old leaves of these mutants. Taken together, these data strongly suggest that NIP6;1 is a boric acid channel required for proper distribution of boric acid, particularly among young developing shoot tissues. We propose that NIP6;1 is involved in xylem–phloem transfer of boric acid at the nodal regions and that the water-tight property of NIP6;1 is important for this function. It is proposed that during evolution, NIP5;1 and NIP6;1 were diversified in terms of both the specificity of their expression in plant tissues and their water permeation properties, while maintaining their ability to be induced under low B and their boric acid transport activities.
Boric acid is a small molecule with the central boron (B) atom possessing three valence electrons. The molecular radius of boric acid is 2.573 Å, similar to those of some other small uncharged molecules such as urea (2.618 Å). Boric acid is a very weak Lewis acid with pKa of 9.24. B in neutral solution is mostly present as boric acid (H3BO3) (reviewed in Marschner, 1995  ; Woods, 1996).
B is an essential element not only for vascular plants but also for diatoms, cyanobacteria, and a number of species of marine algal flagellates (Warington, 1923
One of the primary functions of B in plants is to serve in the cross-linking of rhamnogalacturonan-II (RG-II), a component of cell wall pectic polysaccharides. RG-II is an essential component of a stable three-dimensional pectic network, and borate forms a cross-link with apiose residues of RG-II (reviewed in O'Neill et al., 2001
Symptoms of B deficiency occur mainly in growing or expanding organs in the plant body. Under B-deficient conditions, leaf expansion and root elongation, apical dominance, flower development, and fruit and seed set are inhibited (Marschner, 1995
Recent reports suggest that B can be retranslocated from old tissues to young tissues or can be preferentially transported to sink tissues under B deficiency. Some plant species produce and translocate significant amounts of sugar alcohols, including mannitol and sorbitol. Because sugar alcohols contain cis-hydroxyl groups, they can readily bind to boric acid (forming a poly-B complex) and allow B to be retranslocated through phloem (Brown and Hu, 1996
On the other hand, some sugar alcohol–nonproducing plants, including Arabidopsis thaliana (Noguchi et al., 2000
It has long been believed that B is passively transported; that is, the B transport rate is in proportion to the transmembrane concentration gradients. This was based on the relatively high permeability of boric acid to lipid bilayers, as boric acid is a noncharged molecule (Raven, 1980
However, recent studies revealed the importance of transport proteins in transmembrane B transport (reviewed in Tanaka and Fujiwara, 2008
NIP5;1 belongs to the Major Intrinsic Protein (MIP) family. MIPs are present in mammals, amphibians, yeast, bacteria, and plants and are known to facilitate the passive flow of small uncharged molecules such as glycerol, urea, and formamide as well as water. Thirty-five MIP genes are present in the Arabidopsis genome. Plant MIPs can be subdivided into four distinct groups: the tonoplast intrinsic proteins, the plasma membrane intrinsic proteins (PIPs), the small basic intrinsic proteins, and the nodulin 26 (NOD26)–like intrinsic proteins (NIPs) (reviewed in Johanson et al., 2001
The NIP subfamily is specific for plants. Nine NIP genes are present in Arabidopsis. Some NIPs have been shown to be multifunctional channels that mediate the transport of small uncharged molecules such as glycerol and urea as well as water (reviewed in Wallace et al., 2006
NIP5;1 is a plasma membrane boric acid transporter expressed in root epidermal, cortical, and endodermal cells. Expression of the NIP5;1 transcript is transcriptionally upregulated in response to B deprivation. NIP5;1 is involved in B uptake from the root surface under conditions of B limitation as a major boric acid channel (Takano et al., 2006
It is possible that other members of the NIP family are also involved in B transport. NIP6;1, is the most similar gene to NIP5;1 in Arabidopsis. NIP5;1 and NIP6;1 belong to NIP subgroup II, and computational homology modeling shows that NIP6;1 is predicted to possess a wider pore at the ar/R region compared with NIP subgroup I, and the three-dimensional structure is predicted to be similar to that of NIP5;1 (Wallace and Roberts, 2004
B Transport Activity in Xenopus Oocytes Phylogenetic analysis of the predicted amino acid sequences revealed that among the nine Arabidopsis NIP genes, NIP6;1 is the most similar to NIP5;1 (66.4% amino acid sequence identity and 83.1% similarity) (Wallace and Roberts, 2004 ).
In a previous report, we showed that NIP5;1 transports boric acid efficiently in Xenopus oocytes (Takano et al., 2006 In an osmotic boric acid swelling assay, NIP6;1 and NIP5;1 copy RNA (cRNA)–injected and control oocytes were placed in modified, isoosmotic Ringer's solution (190 mosmol/kg) in which NaCl was replaced with boric acid. Under these assay conditions, boric acid transport into oocytes results in an osmotic gradient that subsequently drives water movement into oocytes, causing an increase in cell volume and oocyte swelling. The initial rate of oocyte swelling is a function of two parameters: the solute permeability for boric acid and the osmotic water permeability (Pf) of the oocyte plasma membrane. NIP6;1- and NIP5;1-expressing oocytes showed significant swelling upon exposure to boric acid, while negative control oocytes (uninjected or mock-injected oocytes) showed little or no swelling under the same conditions (Figures 1A and 1B ; see Supplemental Figure 1A online). In addition, pretreatment of NIP5;1- and NIP6;1-injected oocytes with the aquaporin channel blocker HgCl2 reduced boric acid–induced swelling to levels observed in uninjected oocytes (see Supplemental Figure 1B online). Overall, these properties are consistent with the facilitated transport of boric acid and suggest that NIP6;1 is a boric acid channel, similar to NIP5;1.
The rate of oocyte swelling was slower in NIP6;1-expressing oocytes than in NIP5;1-expressing oocytes (Figure 1A), even though protein gel blot analysis shows a similar expression level of both proteins in oocyte lysates (see Supplemental Figure 2 online). Boric acid uptake was further verified by directly quantifying the amount of boric acid taken up by oocytes using inductively coupled plasma–mass spectrometry (ICP-MS). Figure 1C shows a time course of boric acid uptake for NIP5;1 and NIP6;1 oocytes compared with uninjected controls. Both NIP5;1- and NIP6;1-injected oocytes show linear boric acid uptake rates that are over 50-fold higher than those of uninjected control oocytes (Figures 1C and 1D). To compare the water transport properties of NIP6;1 and NIP5;1, a Pf assay was conducted. The water transport rate of NIP6;1-expressing oocytes was indistinguishable from that of control oocytes, with both showing a low Pf characteristic of diffusion across the bare bilayer. By contrast, oocytes expressing NIP5;1 showed facilitated water transport that was twofold higher than that of control and NIP6;1-expressing oocytes (Figure 2 ). These observations account for the apparent differences in boric acid–induced swelling rate between NIP5;1 and NIP6;1 oocytes. Overall, the transport analyses indicate that both NIP5;1 and NIP6;1 are boric acid transporters but are different in terms of water transport activities, with NIP6;1 forming a water-tight boric acid transport channel.
Subcellular Localization of NIP6;1 Soybean (Glycine max) NOD26, the archetype of the NIP family, is localized on the symbiosome membrane of nitrogen-fixing root nodules (Fortin et al., 1987 ; Weaver et al., 1991), but the subcellular localization data for nonsymbiotic NIPs are limited. Arabidopsis NIP5;1 (Takano et al., 2006) is localized on the plasma membrane, and NIP2;1 is reported to be localized on the plasma membrane (Choi and Roberts, 2007) and the endoplasmic reticulum (Mizutani et al., 2006). To investigate the subcellular localization of NIP6;1, an expression construct (referred to as 35S-GFP-NIP6;1) of NIP6;1 fused to the C terminus of the green fluorescent protein (GFP) under the control of a cauliflower mosaic virus 35S RNA promoter (P35S) was introduced into Arabidopsis plants. Transgenic plants carrying the promoter of NIP5;1 fused to GFP (referred to as Pro-NIP5;1-GFP) were used as a control for subcellular localization of free GFP. NIP6;1-dependent green fluorescence in the root elongation zone and root tips of three independent transgenic lines was observed. Representative images are shown in Figure 3
. GFP fluorescence in cells expressing GFP-NIP6;1 was localized on the cell periphery; by contrast, GFP fluorescence of cells expressing free GFP was detected at the nucleus and cell periphery in the root elongation zone (Figures 3A and 3B).
To exclude the possibility that GFP fluorescence is associated with the tonoplast, transgenic plants were stained with FM4-64 for 5 min and observed after 18 h of incubation in the dark. Under this condition, FM4-64 signals were associated with the tonoplast, while GFP fluorescence remained at the cell periphery of the root tips (Figure 3C), confirming that the GFP florescence is not associated with the tonoplast. To further confirm the plasma membrane localization of NIP6;1, transgenic plants were stained with propidium iodide for cell wall staining and then treated with 0.5 M mannitol solution to induce plasmolysis. GFP fluorescence was separated from the propidium iodide signal at the root elongation zone (Figure 3D), confirming that NIP6;1 is localized to the plasma membrane.
NIP6;1 Transcript Accumulation To test for B-dependent regulation of NIP6;1, quantitative RT-PCR (Q-PCR) was conducted. Wild-type plants (ecotype Col-0) were grown in solid medium containing 100 µM B (+B) for 27 d and then transferred to hydroponic culture solutions containing 100 µM B for 19 d. The plants were then incubated for an additional 24-h period in medium containing either 100 or 0.1 µM B (–B) prior to Q-PCR analysis.
NIP6;1 transcript levels in stems were higher than those in roots and rosette leaves, in contrast with those of the NIP5;1 transcript, which accumulated predominantly in roots (Figure 4
). NIP6;1 mRNA accumulation in stems was increased by 1.4-fold under conditions of B limitation (–B) compared with that under high-B conditions (+B), while the response was not evident in roots and rosette leaves (Figure 4A). NIP5;1 mRNA accumulation in roots was more than sevenfold higher under low-B conditions than that under high-B conditions, as reported previously (Takano et al., 2006
Tissue Specificity of NIP6;1 Expression To analyze the tissue and cell specificities of the expression of NIP6;1, a 2.3-kb promoter fragment upstream of the transcriptional start site fused to the β-glucuronidase (GUS) reporter gene was introduced into Arabidopsis plants. Three independent T3 Pro-NIP6;1-GUS transgenic lines were observed, and representative images are shown in Figure 5 . Strong GUS staining was observed in nodal regions, the base of flowers, and the petioles of immature young rosette leaves, but no staining was observed in mature leaves (Figures 5A and 5B). The expression patterns were identical under both high and low B supply. In transverse cross sections of petioles and nodes, GUS staining was observed in vascular bundles (Figures 5C to 5F).
To further investigate the cell specificity of NIP6;1 expression, an expression construct (Pro-NIP6;1-GFP-NIP6;1) consisting of a cDNA encoding a NIP6;1-GFP fusion protein driven by the promoter NIP6;1 was introduced into Arabidopsis plants. The GFP fluorescence signal was analyzed in 10 independent T1 transgenic lines, with representative images shown in Figure 6 . Cell walls and nuclei were stained with 10 µg/mL propidium iodide. In the cross section of stems, the fluorescence signal of the NIP6;1-GFP fusion protein was most abundant in the region of vascular systems, similar to the case of GUS staining of Pro-NIP6;1-GUS transgenic plants (Figures 5D and 6A). No GFP fluorescence was observed in the wild-type Col-0 plants (Figure 6B). High-magnification images of the vascular bundles demonstrated that NIP6;1 expression is localized on the phloem regions, most likely to the phloem companion cell, phloem parenchyma cell regions, and sieve elements (Figures 6C and 6D). The phloem cells were identified as phloem companion cells and phloem parenchyma cells based on the cells including nuclei and as sieve elements based on the cells including no nuclei.
Plant Growth of NIP6;1 T-DNA Insertion Mutants To characterize the function of the NIP6;1 gene in Arabidopsis, three independent T-DNA insertion mutant alleles for NIP6;1, SM_3_15719, SALK_046323, and SALK_097969, were obtained and named nip6;1-1, nip6;1-2, and nip6;1-3, respectively. nip6;1-1 is from the Exon Trapping Insert Consortium, and nip6;1-2 and nip6;1-3 are from the SALK Institute. Sequence analysis confirmed T-DNA insertion sites in nip6;1-1, nip6;1-2, and nip6;1-3 to be in the first exon, the fourth exon/intron junction, and the 3' untranslated region of the NIP6;1 gene, respectively (Figure 7A ).
To observe growth patterns, the T-DNA insertion lines were grown hydroponically for 28 d supplied with either a low (0.1 µM) or a high (100 µM) B supply. At late vegetative stages, plants supplied with 0.1 µM B showed small young rosette leaves in all three T-DNA insertion lines compared with Col-0 plants (Figure 7B). The expansion of young rosette leaves was strikingly inhibited in these insertion lines. Young rosette leaves were small and dark green in color and irregular in shape in all three nip6;1 mutant plants under 0.1 µM B conditions. By contrast, the mature leaves of nip6;1 mutant plants were apparently normal compared with those of wild-type plants grown under 0.1 µM B conditions (Figures 7B and 7C). These nip6;1 mutant plants grew in a similar manner to the corresponding wild-type plants under high-B concentrations (100 µM B) (Figures 7B and 7C). Because all three nip6;1 mutant plants showed a similar phenotype under high- and low-B conditions, nip6;1-1 and nip6;1-2 mutant plants were used for further experiments. At reproductive stages with 1 µM B supply, loss of apical dominance was observed in nip6;1-1 and nip6;1-2 mutant plants but not in Col-0 plants (Figure 8 ). Under 100 µM B supply, nip6;1-1 and nip6;1-2 mutant plants showed normal growth with apical dominance (see Supplemental Figure 3 online). These experiments were repeated a minimum of three times, and similar results were obtained in each case. These observations demonstrate that NIP6;1 is required for expansion of young rosette leaves and development of the shoot apices under low-B concentrations.
Anatomical Observation of Rosette Leaves When wild-type plants were grown under 0.03 µM B, cell sizes of cauline leaves were reduced and the intercellular air spaces were mostly lost in Arabidopsis (Takano et al., 2001 ). To determine whether nip6;1 mutant plants show a similar developmental defect at low B supply, plants were grown in 1 µM B and the morphology of the young rosette leaves was analyzed (Figure 9
). Sections of young rosette leaves of Col-0 plants showed expanded mesophyll cells and well-developed intercellular air spaces (Figure 9A), whereas young rosette leaves from nip6;1-1 mutant plants showed a smaller cell size and the absence of intercellular air spaces compared with Col-0 plants (Figure 9B). By contrast, both Col-0 plants and nip6;1-1 mutant plants showed well-expanded mesophyll cells and developed intercellular air spaces in old rosette leaves (Figures 9C and 9D). These experiments were repeated two times, and similar results were obtained in each case. These findings suggest that cell expansion in young rosette leaves was inhibited in nip6;1-1 mutant plants under low-B conditions.
B Concentrations in NIP6;1 T-DNA Insertion Mutants To further examine the function of NIP6;1 in B transport, the B contents of mutant and wild-type plants grown under high- and low-B concentrations were determined using ICP-MS. Young and old rosette leaves or stems with shoot apices and rosette leaves were collected from three to four independent plants at vegetative and reproductive stages, respectively. At late vegetative stages, B concentrations in young rosette leaves of nip6;1-1 and nip6;1-2 mutant plants were significantly reduced (26 and 20%, respectively) compared with those of the corresponding leaves in Col-0 plants under low-B growth conditions (5 µM), whereas the B concentrations in the old rosette leaves were not different between the wild type and the insertion lines (Figure 10A ). Under high-B (100 µM) growth conditions, there were no significant differences between B concentrations in young and old rosette leaves of wild-type and nip6;1 mutant plants (Figure 10B).
B concentrations were also measured in stems with shoot apices and rosette leaves during the reproductive stages, and the results were similar to those observed at the vegetative stages (Figure 10C). B concentrations in the stems with shoot apices of nip6;1-1 and nip6;1-2 mutant plants were significantly reduced (  20% in both lines) compared with those of the corresponding stems with shoot apices in Col-0 plants under low-B growth conditions (1 µM B) (Figure 10C). Under growth conditions of high B (100 µM B), there were no significant differences between B concentrations in stems with shoot apices of wild-type and nip6;1 mutant plants (Figure 10D). The B concentrations of rosette leaves of mutant and wild-type plants were indistinguishable, regardless of whether plants were grown in low (Figure 10C) or high (Figure 10D) concentrations. These experiments were repeated three times, and similar results were obtained in each case. These data suggest that NIP6;1 is involved in preferential B transport to sink tissues of shoots (e.g., young rosette leaves) under the limiting B conditions.
B Distribution in Various Tissues in NIP6;1 T-DNA Insertion Mutants In plants supplied with 0.3 µM 10B, 10B uptake into shoot apices was reduced by 27% in nip6;1 mutant plants compared with Col-0 plants (Figure 11A ). By contrast, 10B uptake into rosette leaves was slightly higher in nip6;1-1 mutant plants compared with Col-0 plants. In plants supplied with 100 µM 10B, there was no significant difference in 10B uptake into shoot apices between nip6;1 mutant plants and Col-0 plants (Figure 11B). 10B uptake into rosette leaves was slightly higher in nip6;1-1 mutant plants compared with Col-0 plants at 100 µM 10B supply. These experiments were repeated three times, and similar results were obtained in each case. These findings indicate that B transport into shoot apices is disrupted in nip6;1 mutant plants and provide further support that NIP6;1 is required for preferential B transport to sink tissues of shoots under the conditions of B limitation.
In this study, we demonstrated that NIP6;1 functions as a plasma membrane boric acid channel required for preferential B transport to sink tissues of shoots under B deficiency. NIP5;1 is a boric acid channel required for B uptake in roots and normal growth of Arabidopsis under conditions of B limitation (Takano et al., 2006 ). NIP6;1 is a similar protein to NIP5;1, but NIP6;1 has different physiological roles in B transport from NIP5;1 in Arabidopsis.
NIP6;1 Has Different Transport Properties from NIP5;1 and Forms a Water-Tight Boric Acid Transporter in Xenopus Oocytes Heterologous expression of NIP6;1 in oocytes shows that, similar to NIP5;1, it facilitates rapid uptake of boric acid across the oocyte plasmalemma. Similar to previous observations with other NIP proteins, boric acid transport shows the properties of channel-based facilitated flux (Figure 1), including inhibition by mercurial compounds (see Supplemental Figure 1 online). Subcellular localization analysis of NIP6;1 using transgenic plants showed localization to the plasma membrane (Figure 3). Taken together, the localization and transport data indicate that NIP6;1 is a plasma membrane boric acid channel in plant cells.
Similar to previous reports, analyses in this study show that NIP6;1 is completely impermeable to water (Wallace and Roberts, 2005 From the point of view of their different roles in plant physiology, it is reasonable that NIP5;1 transports water as well as boric acid whereas NIP6;1 facilitates B transport but is water-impermeable. In the case of NIP5;1, expression occurs mainly at the root elongation zone involved in B transport from the root surface into the xylem, along with the pathway of water flow. Thus, in the case of B transport through NIP5;1, it is not necessary to restrict water transport. In contrast with NIP5;1, NIP6;1 promoter activity was observed between xylem and phloem cells at petioles and nodal regions (Figure 5). In nodal regions, GFP-NIP6;1 was observed in phloem regions, including sieve element, phloem parenchyma cells, and companion cells (Figure 6). This localization pattern suggests that NIP6;1 is involved in xylem–phloem transfer of B. If this is the case, NIP6;1 may need to be water-tight because having water-permeable channels in this cell type may disrupt phloem transport.
NIP6;1 Exhibits Different Tissue-Specific and B-Dependent Expression Patterns from NIP5;1 in Arabidopsis GUS staining in 10-d-old Pro-NIP6;1-GUS seedlings was observed in the first nodes and petioles and veins of all leaves (see Supplemental Figure 4 online), while GUS staining in the plants grown for 28 d was observed in the base of flowers and siliques, nodal regions, and veins and petioles of only young rosette leaves (Figure 5). GUS staining in the petioles and veins of young rosette leaves disappeared along with leaf growth, while GUS staining in nodal regions continued to be detected. As we discuss below, these development-dependent expression profiles suggest roles of NIP6;1 in B distribution within young rosette leaves.
NIP6;1 Plays Important Roles in Preferential Transport of B to Sink Tissues for Normal Growth under Conditions of B Limitation in Arabidopsis While expansion of young rosette leaves was inhibited under conditions of B limitation in nip6;1 mutant plants (Figures 7B and 7C), this inhibition was only observed at certain stages of vegetative growth. For example, during the first and second weeks, the growth of nip6;1 mutant plants was apparently normal under low-B conditions (see Supplemental Figure 5 online). Even if facilitated uptake of B is disrupted in nip6;1 mutant plants under low-B conditions (Figure 11A), a smaller but sufficient amount of B molecules might still be taken up into developing tissues by passive diffusion across the lipid bilayer of the cell membrane and other channel proteins. By contrast, the growth of young rosette leaves emerging after several weeks was retarded in nip6;1 mutant plants under low-B conditions (Figure 7). In the presence of fully expanded leaves that can take up large amounts of B through high transpiration rate, facilitated transport of B through NIP6;1 seems to be required to provide enough B for young rosette leaves. Tracer experiments demonstrated that short-term 10B uptake into shoot apices is lower in nip6;1 mutant plants than in the wild-type plants under low-B conditions but not under high-B conditions (Figure 11), suggesting that B is not distributed efficiently to sink tissues in nip6;1 mutant plants. These findings indicate that NIP6;1 is crucial for preferential distribution of B to sink tissues in shoots.
Possible Involvement of NIP6;1 in Xylem–Phloem Transfer under Low-B Conditions
In this study, we provide the following evidence that supports a role of NIP6;1 in the xylem–phloem transfer of B. First, NIP6;1 is expressed in branching points (stem nodal regions) and junction areas (the base of flowers and siliques) (Figure 5), known as places for rapid xylem–phloem transfer (reviewed in Marschner, 1995
Our observations also suggest other roles of NIP6;1. NIP6;1 is expressed strongly in young rosette leaves compared with old rosette leaves (Figure 5; see Supplemental Figure 4 online). Expression in veins of young rosette leaves is not likely to be involved in xylem–phloem transfer of B, as such a process in these tissues is likely to be minimal. NIP6;1 in these tissues may rather be involved in unloading of B from phloem and xylem to facilitate B diffusion to developing leaf mesophyll cells.
Evolutionary and Functional Relationship between NIP5;1, NIP6;1, and Rice NIP3;1 In this study, we demonstrated that NIP6;1 is a boric acid channel involved in preferential B transport to growing tissues of plants and showed the function of a boric acid channel in shoots in Arabidopsis. Transport of B to growing tissues of plants under B-deficient conditions occurs not only by apoplastic flow via the transpiration stream but also via other mechanisms, such as xylem–phloem transfer, which involve facilitated flux across the membranes of living cells. NIP6;1 is involved in this latter mechanism.
Plant Materials Col-0 of Arabidopsis thaliana was derived from our laboratory stocks. Information about the T-DNA insertion lines was obtained from the SIGnAL database (Alonso et al., 2003 ), and the seeds were obtained from the ABRC. The genotypes of plants were determined by PCR using the left border T-DNA–specific primer LBa1 (5'-TGGTTCACGTAGTGGCCATCG-3') and the gene-specific primers nip6;1-1 (5'-TTCCAAATTTTATAAGCATCGTCG-3'), nip6;1-2 (5'-CCCACTTACTCCTCACTCCAACC-3'), and nip6;1-3 (5'-TTCCAAATTTTATAAGCATCGTCG-3' and 5'-ATCCGGCTGTAACCATTGCCT-3').
Plant Growth Conditions
Expression of NIP6;1 and NIP5;1 in Xenopus Oocytes
Osmotic swelling assays for boric acid uptake were conducted by placing the oocytes in isoosmotic Ringer's solution (190 mosmol/kg) with the transport solute (200 mM boric acid) replacing NaCl. The Pf of oocytes was measured at 15°C from the rate of oocyte swelling in hypoosmotic medium (30% Ringer's solution) as described previously (Wallace and Roberts, 2005
The relative expression of NIP5;1 and NIP6;1 in oocytes was assayed by protein gel blot analyses of Xenopus oocyte lysates with an anti-FLAG antibody as described previously (Wallace and Roberts, 2005
Direct B Uptake Measurement in Xenopus Oocytes
Expression of GFP-Tagged NIP6;1 in Arabidopsis Seedlings
A plasmid carrying promoter NIP5;1-GFP was constructed as follows. The region from –2188 to +312 was amplified from BAC clone F24G24 obtained from the ABRC by PCR using primers 5'-GGTGGATCCGAAAGCAAGCATTCCCTG-3' and 5'-GAGCCATGGCCAACGTTTTTTTTTTTGGT-3'. BamHI and NcoI recognition sites are underlined, respectively. The 5' end of full-length cDNA (The Arabidopsis Information Resource database) was used as the predicted transcript start site (+1). The region from –2188 to +312 was excised from the clone using BamHI and NcoI, and the fragment was subcloned into pTF441. pTF441 was constructed from pBI221 (Jefferson et al., 1987
Arabidopsis Col-0 plants were transformed using the floral-dip method (Clough and Bent, 1998
Quantification of Transcript Accumulation by RT-PCR
Histochemical Analysis of NIP6;1
Arabidopsis Col-0 plants were used for plant transformation. The Pro-NIP6;1-GUS transgenic plants (homozygous T3) were grown with hydroponic culture solution containing 30 µM B for 28 d under long-day conditions prior to histochemical analysis. GUS staining was performed as described previously (Shibagaki et al., 2002 The 2355-bp region upstream of the initiation codon of NIP6;1 was amplified from Arabidopsis Col-0 genomic DNA by PCR using the following primers: 5'-GAGCTGCAGGGATTCAAAGATTATGGGA-3' and 5'-AATGGTACCGTCGAGGGTAGAGATAGATG-3'. PstI and KpnI recognition sites are underlined, respectively. The amplified region was excised by digestion with PstI and KpnI, and the fragment was subcloned into the PstI–KpnI sites of the binary vector, 35S-GFP-NIP6;1, containing the GFP-NIP6;1 fragment. The resulting plasmid was termed Pro-NIP6;1-GFP-NIP6;1 as described in Results. The construct was introduced into Arabidopsis plants, and T1 transgenic plants were grown with rockwool and vermiculite for 5 to 6 weeks. Sections of inflorescence stems in the nodal regions were cut by hand using a razor blade, incubated in 10 µg/mL propidium iodide, and analyzed by confocal laser scanning microscopy (LSM 510; Carl Zeiss).
Anatomical Observation of Rosette Leaves
Measurement of B Content in Plants
Accession Numbers
Supplemental Data
The T-DNA insertion lines were kindly provided by the ABRC at Ohio State University. We thank M. Wada, Y. Kawara, and K. Aizawa for excellent technical assistance and K. Miwa and H. Hanaoka for discussion. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to T.F., by a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics for Agricultural Innovation; Grant IPG-0005) to T.F., and by National Science Foundation Grant MCB-0618075 to D.M.R.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Toru Fujiwara (atorufu{at}mail.ecc.u-tokyo.ac.jp).
[W] Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.108.058628 Received February 12, 2008; Revision received September 26, 2008. accepted October 2, 2008.
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