Polar Localization of the NIP5;1 Boric Acid Channel IsMaintained by Endocytosis and Facilitates Boron Transport inArabidopsis Roots

SheliangWang,a,b,c Akira Yoshinari,a,b Tomoo Shimada,d Ikuko Hara-Nishimura,d,e Namiki Mitani-Ueno,f Jian FengMa,f Satoshi Naito,c and Junpei Takanoa,c,1

aGraduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai 599-8531, JapanbGraduate School of Agriculture, Hokkaido University, Sapporo 060-8589, JapancResearch Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, JapandGraduate School of Science, Kyoto University, Kyoto 608-8502, Japane Faculty of Science and Engineering, Konan University, Kobe 658-0072, Japanf Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan

ORCID IDs: 0000-0003-2237-8189 (S.W.); 0000-0001-8814-1593 (I.H.-N.); 0000-0003-3411-827X (J.F.M.); 0000-0001-7006-5609(S.N.); 0000-0002-7474-3101 (J.T.)

Boron uptake in Arabidopsis thaliana is mediated by nodulin 26-like intrinsic protein 5;1 (NIP5;1), a boric acid channel that islocated preferentially on the soil side of the plasma membrane in root cells. However, the mechanism underlying this polarlocalization is poorly understood. Here, we show that the polar localization of NIP5;1 in epidermal and endodermal root cellsis mediated by the phosphorylation of Thr residues in the conserved TPG (ThrProGly) repeat in the N-terminal region ofNIP5;1. Although substitutions of Ala for three Thr residues in the TPG repeat did not affect lateral diffusion in the plasmamembrane, these substitutions inhibited endocytosis and strongly compromised the polar localization of GFP-NIP5;1.Consistent with this, the polar localization was compromised in m subunit mutants of the clathrin adaptor AP2. The Thr-to-Alasubstitutions did not affect the boron transport activity of GFP-NIP5;1 in Xenopus laevis oocytes but did inhibit the ability tocomplement boron translocation to shoots and rescue growth defects in nip5;1-1 mutant plants under boron-limitedconditions. These results demonstrate that the polar localization of NIP5;1 is maintained by clathrin-mediated endocytosis, isdependent on phosphorylation in the TPG repeat, and is necessary for the efficient transport of boron in roots.

INTRODUCTION

Boron is essential for plant growth, and boron deficiency isa worldwide agricultural problem (Shorrocks, 1997). Boron de-ficiency often inhibits root elongation, leaf expansion, and thefertility of crop plants (Marschner, 2012). As borate, boron hasstructural functions in the cell wall, cross-linking pectic poly-saccharides at rhamnogalacturonan II regions (Kobayashi et al.,1996; Ishii and Matsunaga, 1996; O’Neill et al., 1996; Funakawaand Miwa, 2015). In solution, boron is present primarily as boricacid at physiological pH.Boric acid is aweakLewis acidwith apKa

of 9.24 [B(OH)3 + H2O $ B(OH)42 + H+] (Marschner, 2012). Be-

cause boric acid is a small, uncharged molecule, it is transportedacross membranes by passive diffusion (Dordas et al., 2000).However, under low-boron conditions, boron uptake into Arabi-dopsis thaliana root cells is facilitated by NIP5;1, a boric acidchannel localized in the plasma membrane (PM) (Takano et al.,2006). NIP5;1 belongs to the nodulin 26-like intrinsic protein (NIP)subfamily of themajor intrinsic proteins (aquaporins) (Maurel et al.,2015). T-DNA insertion mutants of Arabidopsis NIP5;1 showed

severely reduced root and shoot growth due to defects in boronuptake under low-boron conditions (Takano et al., 2006). Theexpression level of NIP5;1 is repressed by high boron concen-trations through mRNA decay, dependent on its 59-untranslatedregion (UTR) (Tanaka et al., 2011, 2016). These findings estab-lished an essential role for NIP5;1 in boric acid uptake underboron-limited conditions. As the closest paralog of NIP5;1 inArabidopsis, NIP6;1 was identified as a boric acid channel con-tributing to the preferential distribution of boron to young, de-veloping shoot tissues under low-boron conditions (Tanaka et al.,2008). NIP7;1 was also characterized as a boric acid channel andwasexpressed indeveloping anthers (Li et al., 2011). In contrast toNIP5;1 and its homologs, which function as boron importers,BOR1 and its homologs function as boron exporters in the PM(Takanoetal., 2002, 2008). TheArabidopsisborateexporterBOR1is required for boron translocation to the xylem and preferentialdistribution of boron to young, developing shoot tissues (Noguchiet al., 1997; Takano et al., 2001, 2002). A recent electrophysio-logical study of barley (Hordeum vulgare) Bot1, an At-BOR1 ho-molog required for high-boron tolerance in barley, suggested thatHv-Bot1 is a channel-like anion transporter with a high affinity forborate (Nagarajan et al., 2015).Recently, several transport proteinswere shown to be localized

in thePM in apolarmanner, toward the soil or the stele side, in rootcells. At-NIP5;1 is localized on the soil-side PM domain of theoutermost root cell layers under control of the NIP5;1 promoter

1 Address correspondence to jtakano@plant.osakafu-u.ac.jp.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Junpei Takano (jtakano@plant.osakafu-u.ac.jp).www.plantcell.org/cgi/doi/10.1105/tpc.16.00825

The Plant Cell, Vol. 29: 824–842, April 2017, www.plantcell.org ã 2017 ASPB.

(Takano et al., 2010) and in various root cells, including endo-dermal cells, when expressed ectopically (Alassimone et al.,2010). In contrast, At-BOR1 and At-BOR2 are localized on thestele-side PM domains of various root cells, including the epi-dermis and endodermis (Takano et al., 2010; Miwa et al., 2013;Yoshinari et al., 2016). The rice (Oryza sativa) silicic acid channelOs-Lsi1/NIP2;1 (Ma et al., 2006), the auxin precursor indole-3-butyric acid transporter At-ABCG37/PIS1/PDR9 and its homologAt-ABCG36/PEN3/PDR8 (Strader andBartel, 2009;Ruzicka et al.,2010), the potato (Solanum tuberosum) phosphate transporterSt-PT2 (Gordon-Weeks et al., 2003), the maize (Zea mays) ferric-phytosiderophore transporter Zm-YS1 (Ueno et al., 2009), themetal transporter Os-Nramp5 (Sasaki et al., 2012), the nitratetransporter At-NRT2.4 (Kiba et al., 2012), and the iron transporterAt-IRT1 (Barberon et al., 2014) are localized toward the soil sideor rootcells.ThemanganeseexporterOs-MTP9 (Uenoetal., 2015)and the silicate exporter Os-Lsi2 (Ma et al., 2007) are localizedtoward the stele side. These examples suggest the generalimportance of polar localization toward the soil or stele side forthe radial transport of nutrients and other substances in rootsand indicate the existence of common mechanisms for suchpolar localization. Os-Lsi1, Os-Lsi2, Os-Nramp5, and Os-MTP9show polar localization in the exodermis and endodermis, whereCasparian strips restrict both solute diffusion in the apoplastand lateral diffusion of proteins in the PM (Alassimone et al.,2010; Barberon and Geldner, 2014). The other transportproteins, including At-NIP5;1, show polar localizations in cellswithout Casparian strips. These observations suggest that dy-namic mechanisms are required to establish and maintain polarlocalization.

Mechanisms for the polar localization of PIN-FORMED auxinefflux carriers (PINs) have been extensively studied. PIN1 is lo-calized to the basal (rootward) PM domain of the stele and en-dodermal cells in roots (Friml et al., 2004;Kleine-Vehn et al., 2008).PIN2 is localized to theapical (shootward)PMdomainofepidermalcells and basal PM domain of cortical cells in the root tip region inArabidopsis (Friml et al., 2004). Maintenance of the polar locali-zation of PINs in the PM has been attributed to reduced lateraldiffusion constrained by the cell wall, constitutive recycling, im-mobilized clustering, and spatially defined endocytosis (Feraruet al., 2011; Kleine-Vehn et al., 2011). The direction of intracellulartrafficking is dependent on the function of PID/WAG1/WAG2kinases and PP2A/FyPP1/FyPP3 phosphatases, which act an-tagonistically in reversible phosphorylation in the central hydro-philic loopofPINs (Ganguly et al., 2012; Löfke et al., 2013;OffringaandHuang, 2013; Kania et al., 2014). A high phosphorylation stateof PIN1 and PIN2 recruits them to the apical PM domain, whereasa low phosphorylation level recruits them to the basal PM domain(Kleine-Vehn et al., 2008, 2009). The recycling of PINs to the basalPM domain is dependent on the brefeldin A (BFA)-sensitive ADP-ribosylation factor-guanine nucleotide exchange factor (ARF-GEF) GNOM that activates small GTPases of the ARF class tomediatevesiclebudding in thesecretionpathway (Naramotoetal.,2014; Doyle et al., 2015).

Compared with the mechanisms underlying the polar locali-zation of PINs in the apical-basal direction, little is knownabout polar localization in the radial direction. For At-BOR1,endocytic recycling, which is dependent on DYNAMIN-RELATED

PROTEIN1s, clathrin adaptor proteins, and BFA-sensitive ARF-GEFs, has been implicated in polar localization toward the steleside (Takano et al., 2010; Wakuta et al., 2015; Yoshinari et al.,2016). The polar localization of At-ABCG36 and At-ABCG37 to-ward the soil sidewas not related to the functions of components/regulators of apical-basal trafficking of PINs because polar lo-calizationwasmaintained ingnom,axr4, van7,pinoid, andpp2aa1pp2aa2 mutants (Łangowski et al., 2010).The polar localization of importers toward the soil side and

exporters toward the stele side in the PM of the epidermal andendodermal cells would be expected to contribute to the efficientuptake and translocation of nutrients in the roots. Amathematicalmodel of silicon uptake in rice roots with various localizationpatterns of the Os-Lsi1 channel and the Os-Lsi2 exporter pre-dicted that the highest silicon uptakewould be generatedwith thelocalization pattern found in the wild-type rice root (Sakurai et al.,2015). However, because of a lack of appropriate experimentalmaterials, the physiological significance of the polar localizationfor nutrient transport has not been demonstrated sufficiently. Theiron transporter At-IRT1was shown to accumulate in the soil-sidePM domain under metal-depleted conditions (Barberon et al.,2014). The polar localization of IRT1 became nonpolar follow-ing the overexpression of FYVE1, a phosphatidylinositol-3-phosphate binding protein recruited to late endosomes. Thehomeostasis of iron and other metals was impaired in FYVE1-overexpressing lines, suggesting that polar localization of IRT1 isimportant for the transport of metals (Barberon et al., 2014).However, it cannot be ruled out that the overexpression of FYVE1affected other processes involved in metal homeostasis.In this study, we showed that polar localization of NIP5;1 was

mediated byThr phosphorylation in a conserved three-amino acid(ThrProGly [TPG]) repeat that is found in its N-terminal region. Ourpharmacological and genetic analyses also provide evidence thatclathrin-mediated endocytosis was dependent on this phos-phorylation and was required for maintenance of the polar lo-calization of NIP5;1. Furthermore, by expressing GFP-NIP5;1wild-type protein and its weak-polar variant in a T-DNA insertionmutant ofNIP5;1, we established the physiological importance ofthepolar localizationofNIP5;1 inboron transport andplantgrowthunder boron-limited conditions.

RESULTS

NIP5;1 and NIP6;1 Show Polar Localization in the PlasmaMembrane of Root Cells

To identify amino acid residues required for the polar localizationof NIP5;1, we first compared the localization of NIPs in Arabi-dopsis. Previously, NIP5;1 (Takano et al., 2006) and NIP6;1(Tanaka et al., 2008) were reported to be localized primarily in thePM, whereas NIP1;1 and NIP2;1 were located both in the PM andin intracellular membranes, most likely the endoplasmic reticulum(Geldner et al., 2009; Mizutani et al., 2006; Choi and Roberts,2007). To compare localization in the same cell types without theinfluence of native NIP5;1, GFP-fused proteins were expressedunder control of the NIP5;1 promoter in the nip5;1-1 mu-tant throughout this study. In epidermal cells, GFP-NIP1;2,

Polar Localization of NIP5;1 for Boron Transport 825

GFP-NIP5;1, and GFP-NIP6;1 were localized mainly in the PM(Supplemental Figure 1). GFP-NIP1;1, GFP-NIP2;1, and GFP-NIP4;1 were localized in both the PM and intracellular compart-ments, presumably in endoplasmic reticulum membranes, andGFP-NIP3;1 and GFP-NIP7;1 were localized in intracellularcompartments (Supplemental Figure 1).

We next investigated whether GFP-NIP1;2 and GFP-NIP6;1showedpolar localizations in thePMby comparing their signalswith those of FM4-64, a lipophilic styryl dye considered to stainthe PM in a nonpolar manner (Malínská et al., 2014; Ueharaet al., 2014; Wakuta et al., 2015). The GFP signals of GFP-NIP5;1 and GFP-NIP6;1 were clearly polarized toward the soilside in epidermal cells (Figure 1A). By contrast, the localizationof GFP-NIP1;2 showed only a weakly polar localization in theoptical longitudinal section. Signal intensities from differentpositions in the longitudinal optical section are affected by thereduction of light penetration of the tissue. It should also benoted that the degree of reduction is dependent on wave-length. Indeed, when PMmarkers YFP-NSPN12 and mCherry-NPSN12were expressed under control of theUBQ10 promoterin the same root, YFP signals showed slightly more polar-ized distribution toward the soil side than mCherry signals(Supplemental Figure 2A).

Therefore, we also used cross sections to evaluate polar lo-calization (Figure 1A). Again, the localizations of GFP-NIP5;1 andGFP-NIP6;1 were clearly polar, whereas that of GFP-NIP1;2 wasweakly polar. To evaluate the polar localization quantitatively, wedefined the polarity index (PI) as follows. In longitudinal opticalsections of Arabidopsis roots, the PI represents the ratio of GFPsignals normalized by FM4-64 signals of the soil-side half versusthe stele-side half (Figure 1C, PILongitudinal) (Wakuta et al., 2015). Inthe cross sections, the PI represents the ratio of GFP signal in-tensity of the soil-side versus the stele-side portions (Figure 1D,PICross). Therefore, a PI of 1.0 represents a nonpolar localizationwith an equal amount of protein in the two halves/portions. ThePILongitudinal values for GFP-NIP5;1 and GFP-NIP6;1 were ;3.8and;3.3, and those of PICross were;11.5 and;5.4, respectively(Figures 1C and 1D), showing preferential localization on theoutermost, soil-facing side of the root cells. By contrast, thePILongitudinal value for GFP-NIP1;2 was;1.5 and the PICross valuewas;2.0 (Figures1Cand1D). Thesevalueswere lower than thosefor GFP-NIP5;1, but still >1.0, representing a weak polar locali-zation favoring the soil side.

NIP1;2, PIP2;1, NPSN12, and LTI6a Show Weak PolarLocalization toward the Soil Side of Root Epidermal Cells

To examine whether this weak polar localization of NIP1;2 iscommon for membrane proteins in the PM of epidermal cells,we observed and quantified the localization of several PMmarkers normally expected to be nonpolar. For this purpose,GFP-tagged LTI6a, a small hydrophobic protein with twopotential transmembrane domains (TMDs) (Cutler et al., 2000);GFP-tagged NPSN12, a plant-specific SNARE with one TMD(Geldner et al., 2009); andGFP-taggedPIP2;1, awater channel(Zwiewka et al., 2015), were expressed in the nip5;1-1 mutantunder the control of theNIP5;1 promoter. As with GFP-NIP1;2,thesePMmarkers showed reduced signals from the soil side to

the stele side in the transverse PM domain compared with theFM4-64 signals and the PILongitudinal values were;1.5 (Figures1A and 1C). In cross sections, intensities of the GFP signalwere higher in the soil-side PMdomain and PICross values were1.3 to 2.5 (Figures 1A and 1D), values that were similar to thoseof GFP-NIP1;2. The GFP-LTI6a and GFP-NPSN12 fusionsshowed weak expression in the cortical cells even under thecontrol of the NIP5;1 promoter. Thus, the PICross values ofGFP-LTI6a and GFP-NPSN12 in the epidermal cells are likelyunderestimates. In addition to GFP-fused PM markers,mCherry-NPSN12 under control of the UBQ10 promoter andPIP2;1-mCherry under control of the CaMV 35S promoter alsoshowedweakly polar localization in epidermal cells in the crosssection (Supplemental Figures 2B and 2C). These resultssuggest that weak polar localization toward the soil side is aninherent feature of membrane proteins on the PM of the epi-dermal cells. For simplicity, we classify GFP-NIP1;2, GFP-PIP2;1, GFP-LTI6a, and GFP-NPSN12 as general PMmarkersin this study.

The N-Terminal Region of NIP5;1 Is Required forPolar Localization

Considering thehigh sequence similarity in the central region fromthe first to the sixth TMDs and the lower conservation in the cy-tosolic N- and C-terminal regions among the NIPs (SupplementalFigure 3A), wehypothesized that theN- or theC-terminal region ofNIP5;1 is probably responsible for the polar localization. To testthis, we generated three chimeric constructs by swapping thecorresponding N-terminal region (Nt), C-terminal region, and theTMD-containing central region from NIP5;1 and NIP1;2, andtagged themwith GFP at the N terminus (Figure 1B). The chimeraGFP-NIP5;1-Ct1;2, in which the C-terminal region of NIP5;1 wasreplaced with that of NIP1;2, showed PILongitudinal and PICross

values of 2.1 and 4.4, respectively (Figures 1C and 1D). Thechimera GFP-Nt5;1-TMD1;2-Ct5;1, in which the TMD-containingcentral region of NIP5;1 was replacedwith that of NIP1;2, showedPILongitudinal andPICross values of 2.3 and 7.2, respectively (Figures1C and 1D). These values were significantly higher than those forGFP-NIP1;2 (P < 0.01, Student’s t test). By contrast, the chimeraGFP-Nt1;2-NIP5;1, in which the Nt of NIP5;1 was replaced withthat of NIP1;2, was localized in the PM in a manner similar to thegeneral PM markers (Figure 1B), with PILongitudinal and PICross

values of 1.5 and 2.1, respectively (Figures 1C and 1D). Theseresults indicated that theN-terminal regionofNIP5;1was requiredfor the polar localization.We then investigatedwhether theN-terminal regionofNIP5;1

was sufficient to confer polar localization on either its homologNIP1;2 or the water channel PIP2;1. For this, we generatedchimeric constructs by replacing the Nt region of NIP1;2 orPIP2;1with that ofNIP5;1, andwe then tested the localizationofthe respective proteins in the nip5;1-1 mutant (Figure 1B). TheGFP-Nt5;1-NIP1;2, inwhichNt ofNIP1;2was replacedwith thatof NIP5;1, showed a clear polar distribution, with PILongitudinal

and PICross values of 2.1 and 8.4, respectively (Figures 1B to1D). Likewise, theGFP-Nt5;1-PIP2;1, in which Nt of PIP2;1 wasreplaced with that of NIP5;1, also displayed clear polar local-ization, with PILongitudinal and PICross values of 2.7 and 9.4,

826 The Plant Cell

Figure 1. The N-Terminal Region of NIP5;1 Is Important for Polar Localization.

(A) and (B)Evaluationof polar localization of proteinsN-terminally taggedwithGFP in root epidermal cells. FM4-64wasused to stain thePMand is shown inmagenta.GFPsignals in thecrosssectionsarecolor-coded from0 (black) to255 (white)with ImageJsoftware to indicate relativeabundance.Bars=20mminlongitudinal sections and 50 mm in cross sections.

Polar Localization of NIP5;1 for Boron Transport 827

respectively (Figures 1B to 1D). These values were significantlyhigher than those of GFP-NIP1;2 or GFP-PIP2;1 (P < 0.01,Student’s t test), indicating that the N-terminal region ofNIP5;1 is sufficient to establish polar localization of aquaporinhomologs.

The Conserved TPG Repeat Is Required for PolarLocalization of NIP5;1

Although the amino acid sequences of the N-terminal regions ofArabidopsis NIPs vary (Supplemental Figure 3A), there is a char-acteristic pattern in NIP5;1 and NIP6;1 that is not present in otherNIPs inArabidopsis: theTPGTPGTPGsequenceatpositions18 to26 of NIP5;1 and the TPGTPG sequence at positions 16 to 21 ofNIP6;1 (Figure 2A). Os-NIP3;1 and Zm-NIP3;1, the rice andmaizeorthologs of Arabidopsis NIP5;1, respectively, are boric acidchannels (Durbak et al., 2014; Leonard et al., 2014; Hanaoka et al.,2014). Alignment of their N-terminal regions showed that theyshare the TPGTPG/A sequence (Supplemental Figure 3B).TPGTPG/A or similar sequences in the N-terminal region wereidentified in AtNIP5;1 homologs in various plant species (Wallaceet al., 2006; Bienert et al., 2008).

To test the importance of this conserved sequence in polarlocalization, we analyzed the localization of GFP-NIP5;1 var-iants in which 17 (ΔNt1–17) or 26 (ΔNt1–26) amino acids fromthe N terminus were deleted. The GFP-NIP5;1ΔNt1–17 variant,which retains the TPG repeat, showed polar localization (Figure2B), although PIs were lower than that of the wild-type con-struct (Figure 2E). TheGFP-NIP5;1ΔNt1–26variant,whichdoesnot contain the TPG repeat, showed significantly lower PIvalues than those of the wild type and the ΔNt1–17 variant(Figures 2B and 2E), although the PI values were a little higherthan those of general PM markers (Figure 1). These resultsshow that the TPG repeat is required for the polar localizationof NIP5;1. The GFP-NIP5;1ΔNt1–17 and GFP-NIP5;1ΔNt1–26variants were observed in punctate structures in cells in ad-dition to the PM (Supplemental Figures 4B and 4C). To testwhether a significant proportion of the variantswas transportedto endosomes and the vacuole, we imaged the cells after darktreatment. In the dark, GFP in the vacuole is relatively resistantto degradation (Tamura et al., 2003). Following a dark treat-ment for 8 h, a large fraction of GFP-NIP5;1ΔNt1–17 andGFP-NIP5;1ΔNt1–26 variants was observed in the vacuole(Supplemental Figures 4E and 4F), suggesting that theN-terminal 17 amino acid sequence is important for retainingthe protein in the PM.

NIP5;1 Is Phosphorylated at the Threonine Residue(s) in theTPG Repeat

We focused on the Thr residues in the conserved TPG repeatbecause these have been predicted to be phosphorylation sites(Wallace et al., 2006; Bienert et al., 2008). To investigate phos-phorylation of these residues, a polyclonal antibody was gener-ated against an artificial peptide of the NIP5;1 N-terminal regioncontaining three phospho-Thr residues (T18/T21/T24). GFP-NIP5;1 variants, in which either one or all three Thr residues atpositions 18, 21, and/or 24 (Figure 2A) were substituted by Ala,were expressed in the nip5;1-1mutant under control of theNIP5;1promoter. For immunoblotting, we prepared immunoprecipitatedsamples containing GFP-NIP5;1 wild type and variants to reducenonspecific binding. Using an anti-GFP antibody, GFP-NIP5;1wild type and variants were detected as bands at ;50, ;100,;130,;190, and;270 kD (Figure 2C). Considering the predictedmolecular mass of GFP-NIP5;1 (58.5 kD) and that the functionalform of aquaporins is a tetramer (Maurel et al., 2015), we suggestthat these bands include monomer, dimer, trimer, tetramer, andunknown modified forms. Using the phospho-Thr NIP5;1 anti-body, these formsofwild-typeGFP-NIP5;1proteinweredetected,whereas those of the T18A$T21A$T24A variant were not (Figure2C). Thewild-type signals disappeared after lambdaphosphatasetreatment (Figure 2C), indicating that the antibody recognizedGFP-NIP5;1 in which the TPG repeat is present in a phosphory-lated state. The GFP-NIP5;1 T18A and T24A proteins were de-tected prominently, but less efficiently than wild-type proteins,whereas the T21A variants were not. These results indicate thatNIP5;1 is phosphorylated at conserved Thr residue(s) in vivo andthat Thr-21 is especially important in this respect. It should benoted that this approach does not unambiguously determine thephosphorylated Thr residue(s) in the TPG repeat in the wild typebecause Ala substitution has the potential to affect the phos-phorylation of neighboring Thr residues.

Threonine Phosphorylation in the TPG Repeat Mediates thePolar Localization of NIP5;1

To assess whether Thr residue phosphorylation was involved inthe polar localization of NIP5;1 in the PM, we investigatedboth the localization of the phosphomimetic mutant GFP-NIP5;1T18D$T21D$T24D, in which Thr residues at positions 18, 21,and 24 were replaced with Asp, and the localization of thenonphosphorylatable mutant GFP-NIP5;1 T18A$T21A$T24Ain epidermal cells. The localization pattern of GFP-NIP5;1

Figure 1. (continued).

(A) Polar localization of NIPs (NIP5;1, NIP6;1, and NIP1;2) and PM markers (LTI6a, NPSN12, and PIP2;1).(B) Polar localization of chimeric proteins. Colors in illustrations to the left represent protein domains used for the chimeras: green, NIP5;1; yellow, NIP1;2;magenta, PIP2;1. Ct, C-terminal region.(C) PIs in optical longitudinal sections. PIs were calculated as signal ratios of GFP relative to FM4-64 on the soil- and stele-side halves of the transverse(apical/basal) PMdomain in the optical longitudinal section (PILongitudinal). n= a total of 30 to 50 epidermal cells from3 to 5 independent T1 transgenic plants.Means 6 SD are shown. Dashed line indicates a PI of 1 (nonpolar).(D) PIs in lateral PM domains. PIs were calculated as relative signal ratios of GFP in the outer- and innermost portions of the lateral PM domains (PICross).Green, location of GFP fusions in the plasmamembrane; white rectangles, 2 mm3 2-mm regions used to calculate PIs. n = a total of 30 to 60 epidermal cellsfrom 5 to 10 independent transgenic lines. Means 6 SD are shown. Dashed line indicates a PI of 1 (nonpolar).

828 The Plant Cell

Polar Localization of NIP5;1 for Boron Transport 829

T18D$T21D$T24D was similar to that of wild-type GFP-NIP5;1(PILongitudinal value, 3.4; PICross value, 9.0; Figures 2D and 2E),whereas GFP-NIP5;1 T18A$T21A$T24A showed amarked loss inpolar localization (PILongitudinal value, 1.5;PICross value, 2.1; Figures2D and 2E). These results indicate the importance of the Thrresidues in the TPG repeat for the polar localization.

We next investigated the effects of substitution in cell typesother than epidermal cells. In transgenic plants with relativelystrong expression, we detected the expression of GFP-NIP5;1 inmature endodermal cells (Figure 2F). As reported previously, theFM4-64 signal was localized exclusively on the soil-side PMdomain due to blocked diffusion in both the apoplast and the PMby the Casparian strips (Figure 2F; Alassimone et al., 2010). Wild-type GFP-NIP5;1 was observed only on the soil-side PM domain,decorated with FM4-64, whereas the T18A$T21A$T24A variantwas observed in both the soil-side and the stele-sidePMdomainsin the endodermis (Figure 2F). Imaging of the cross sectionsupported this. These results suggested a common mechanismregulating the polar localization of NIP5;1 in epidermal and en-dodermal cells, independent of the presence of a diffusion barrier(Casparian strips) in the PM. We also investigated the localizationof thesingleAla-substitutedvariants, T18A,T21A,andT24A.EachAla substitution affected polar localization to a different degree(Figures 2D and 2E). The T21A variant showed the lowest PI, al-though still higher than the T18A$T21A$T24A triple substitutionvariant. Togetherwith the finding that T18AandT24Avariants, butnot the T21A variant, were detected by the phospho-Thr NIP5;1antibody (Figure 2C), these results suggest a correlation betweenphosphorylation status and the degree of polar localization.

To examine whether Thr residue phosphorylation was alsoimportant for the polar localization of NIP6;1, we focused on T15,T16, and T19, residues predicted to be putative phosphorylationsites according to the PhosPhAt database (Zulawski et al., 2013).In contrast to the polar localization of wild-type GFP-NIP6;1, theT15A$T16A$T19A variant lost its polar localization (SupplementalFigure 5A). The PILongitudinal and PICross values were 1.3 and 1.6(Supplemental Figure 5B), which were similar to those obtainedwith the general PM markers (Figures 1B and 1C). Similar to the

case of NIP5;1, single substitutions of these Thr residues (atpositions 15, 16, and 19) with Ala affected the polar localization ofGFP-NIP6;1 to differing extents (Supplemental Figures 5A and5B). These findings indicate the common importance of con-served Thr residues in the TPG repeats for polar localization ofNIP5;1 and NIP6;1 in the PM.Next, we investigated time-dependent changes in the localization

of newly synthesized GFP-NIP5;1 under control of the NIP5;1 pro-moterandthe59-UTRbytransferringplants fromhigh (100mM) to low(0.3 mM) boron conditions (Supplemental Figure 6). The 59-UTR ofNIP5;1 helps to regulate mRNA degradation, depending on boronconditions (Tanaka et al., 2011, 2016). The signals of wild-typeGFP-NIP5;1 in the PM were detectable after 1.5 h under low-boronconditions and the pattern was already polar at this point(SupplementalFigure6).The intensityofwild-typeGFP-NIP5;1onthesoil side, butnoton thesteleside, increasedcontinuously for 24h.Bycontrast, the intensityofGFP-NIP5;1T18A$T21A$T24A increasedonboth sides and showed consistently weak polar localization for 24 h(Supplemental Figure 6). These results support our findings thatphosphorylation of the Thr residues in the conserved TPG repeat isimportant in establishing the polar localization of NIP5;1.

Threonine Phosphorylation Does Not Affect LateralDiffusion of NIP5;1 in the PM

Polar localization in the PM can be generated and maintainedby polar trafficking, including secretion and endocytic recy-cling, and/or retention in specific membrane domains. To ex-amine the effects of phosphorylation on the dynamics ofNIP5;1within and around the PM, we performed fluorescence re-covery after photobleaching (FRAP) analyses on the soil-sidePM surface of root epidermal cells. To inhibit de novo synthesisof GFP-NIP5;1, we applied cycloheximide (CHX; 50 mM) at30 min prior to the FRAP analyses. We confirmed that CHXtreatment inhibited the new synthesis of GFP-NIP5;1 effec-tively (Supplemental Figure 7). In the FRAP analysis, wild-typeGFP-NIP5;1 and the T18A$T21A$T24A variant showed com-parable fluorescence recovery up to 30 min (Figure 3A). We

Figure 2. Phosphorylation of Threonine Residues in the TPG Repeat Mediates Polar Localization of NIP5;1.

(A)ConservedTPGrepeats in theNt regionsofNIP5;1andNIP6;1.Numbers represent thepositionsofThr residueshighlighted inpurple in theproteincodingsequence.(B)Confocal imagesofGFP-NIP5;1DNt1–17andGFP-NIP5;1DNt1–26 in epidermal cells. Bars=20mmin longitudinal sections and50mmin cross sections.The relative fluorescence intensities of GFP signals are color-coded from 0 (black) to 255 (white) with ImageJ software.(C) Immunoblot (IB) analyses of wild-typeGFP-NIP5;1 and variantswith Thr-to-Ala substitution(s) to detect phosphorylation. Immunoprecipitated sampleswith microbeads conjugated with an anti-GFP antibody (a-GFP) were treated with or without lambda phosphatase for in vitro dephosphorylation. Im-munoblotting was performed with an anti-phospho-Thr NIP5;1 antibody (a-pT), which was raised against a phosphorylated NIP5;1 peptide. The samemembrane was reprobed with the anti-GFP antibody to visualize GFP-NIP5;1 variants.(D) Localization of GFP-NIP5;1 variants with single or triple Thr substitutions in epidermal cells. Bars = 20 mm in longitudinal sections and 50 mm in crosssections.(E)PIsofproteins inoptical longitudinal sections (PILongitudinal) andcrosssections (PICross). Values representmeans6 SD,n=a total of30 to50epidermal cellsfrom3 to 5 independent transgenic T1plants for PILongitudinal and n= a total of 30 to 60 epidermal cells from5 to 10 independent transgenic plants for PICross.PIs of GFP-NIP5;1 are the same as in Figure 2A. Dashed lines indicate a polarity index of 1 (nonpolar). Different letters above each bar indicate a significantdifference (one-way ANOVA with LSD-Duncan’s test, P < 0.01).(F) Localization of wild-type GFP-NIP5;1 and T18A$T21A$T24A proteins in mature endodermal cells. White arrows indicate the localization of GFP inendodermal cells, and yellow arrowheads indicate the Casparian strips. co, cortex cells; en, endodermal cells; ep, epidermal cells. Bars = 50 mm inlongitudinal sections and cross sections.

830 The Plant Cell

then examined the fluorescence recovery at the centerand periphery of the bleached area. The rates of recovery at30 min were significantly lower at the center than at theperiphery for both GFP-NIP5;1 and T18A$T21A$T24A proteins(Figure 3B). These results suggest that lateral diffusion con-tributed significantly to fluorescence recovery of the wild-type

GFP-NIP5;1 and T18A$T21A$T24A proteins under these ex-perimental conditions.To further compare the lateral diffusionofwild-typeGFP-NIP5;1

and the T18A$T21A$T24A variant, we used sodium azide (NaN3;0.02%) together with CHX (50 mM) for 30 min prior to FRAPanalysis.NaN3blockshydrolysisby themitochondrialATPaseand

Figure 3. Threonine Residues in the TPG Repeat Are Not Involved in Lateral Diffusion of NIP5;1 in the PM.

(A) and (B)FRAPanalysis ofwild-typeGFP-NIP5;1 and theT18A$T21A$T24Amutant on the soil-side surface of epidermal cells. PlantswerepretreatedwithCHX (50 mM) for 30 min.(A) Time course following photobleaching. Images taken either before (prebleach), immediately after (<1 min), and 5, 15, and 30min after photobleaching.Bar = 20 mm.(B) Fluorescence recovery at the center, periphery, and total ROI at 30 min after photobleaching. Values represent means6 SD. n = 20 to 21 ROIs from fiveroots. Asterisks indicate a significant difference between the center and the periphery of the bleached ROIs (Student’s t test, P < 0.01).(C) Inhibition of internalization of FM4-64. Sodium azide (0.02%) was applied 30 min prior to imaging. Bar = 20 mm.(D) FRAPanalysis using sodiumazide to analyze lateral diffusion in thePM. The diagramshows the definition of region X (donut ring) and region Y (bleachedregion). The areas of X and Y are the same. Values represent means 6 SD. n = 12 to 22 ROIs from 4 to 6 roots.

Polar Localization of NIP5;1 for Boron Transport 831

thus is used as an energy inhibitor. Under these conditions, en-docytosis of FM4-64 was inhibited effectively, compared withthe control condition (Figure 3C). To estimate the rate of lateraldiffusion precisely at the PM surface, changes in the signal ratiosfor a region around (X) and precisely at the bleached region (Y) inthe presence of CHX alone or in combination with NaN3 werecalculated (Figure 3D). The signal ratio, X/Y, was not affectedsignificantly by NaN3 treatment and showed no significant dif-ference at any time point between the wild-type GFP-NIP5;1and T18A$T21A$T24A proteins (Figure 3D). These results in-dicated both that the rate of lateral diffusion in the PM was sim-ilar for the wild-type GFP-NIP5;1 and T18A$T21A$T24A proteinsand that the impact of recycling is of little significance, at leastwithin 30 min. Thus, it seems likely that phosphorylation of theThr residues in the TPG repeat is not involved in retention ina PM domain.

Threonine Phosphorylation Accelerates Endocytosisof NIP5;1

To assesswhether Thr residue phosphorylationwas involved invesicle trafficking, we used BFA, an inhibitor that enables thevisualization of internalized PM proteins by inhibiting theirrecycling to thePM (Geldner et al., 2003;Naramoto et al., 2014).To reduce accumulation of de novo synthesized proteins, wetreated roots with CHX (50 mM) for 30 min and then with BFA(50 mM) and CHX (50 mM) for 60 min. Consistent with a previousreport, BFA caused the formation of aggregates containingGFP-NIP5;1 in root epidermal cells (Figures 4A and 4B; Takanoet al., 2010). The rate of intracellular accumulation (Yoshinariet al., 2016) of GFP-NIP5;1 protein was increased, from 6.9 to12.8%, by BFA treatment in the presence of CHX (Figure 4E).Pretreatment with CHX did not decrease the accumulation of

Figure 4. Threonine Residues in the TPG Repeat and AP2 Are Required for Endocytosis of NIP5;1.

(A) to (C) Localization of wild-type GFP-NIP5;1 (WT), T18A$T21A$T24A, and T18D$T21D$T24D in root epidermal cells. Roots were incubated with CHX(50 mM) and DMSO (0.1%) or BFA (50 mM), as indicated. Bar = 20 mm.(D) Localization of GFP-NIP5;1 in the ap2m mutant roots treated with CHX and BFA, as indicated. Bar = 20 mm.(E) Quantification of rates of intracellular accumulation of GFP-NIP5;1 variants. n = a total of 35 to 50 cells from 3 to 4 roots. Means 6 SD are shown.

832 The Plant Cell

GFP-NIP5;1 significantly in cells compared with treatment withBFA alone (Figures 4C and 4E, P < 0.01, Student’s t test),suggesting that GFP-NIP5;1 in the aggregates is transferredmainly from the PM. By contrast, the T18A$T21A$T24A variantaccumulated little in the BFA-induced aggregates (Figures 4Band 4E), whereas GFP-NIP5;1 T18D$T21D$T24D was accu-mulated in the aggregates and the rate of intracellular accu-mulation increased, from 8.1 to 12.2%, by BFA treatment(Figures 4B and 4E). These results suggest that phosphory-lation of the Thr residues in the TPG repeat accelerated theendocytosis of NIP5;1.

Clathrin-Mediated Endocytosis Is Required for theMaintenance of Polar Localization of NIP5;1 in the PM

Clathrin-mediated endocytosis is a major endocytic pathway inArabidopsis (Fan et al., 2015; Paez Valencia et al., 2016). A het-erotetrameric adaptor protein complex, AP2, which is conservedin eukaryotes, is important for clathrin-mediated endocytosis inArabidopsis (Bashlineet al., 2013;Fanetal., 2013;Kimetal., 2013;Yamaoka et al., 2013). To determine whether clathrin-mediatedendocytosis contributed to the polar localization ofGFP-NIP5;1 inthe PM, we investigated the localization of GFP-NIP5;1 in T-DNAinsertion mutants of the m subunit of the AP2 complex (AP2M). Intheap2m-1andap2m-2mutants,GFP-NIP5;1didnot accumulatesignificantly in BFA-induced aggregates in root epidermal cells(Figures 4D and 4E), indicating a low rates of endocytosis. Thepatterns of GFP-NIP5;1 localization in the epidermal cells ofap2m-1 and ap2m-2 mutants showed a strongly compromisedpolar distributionwithPILongitudinal values of 1.6 and2.0 andPICross

values of 2.8 and 2.7, respectively (Figure 5; SupplementalFigure 8). These results demonstrate that clathrin-mediated

endocytosis is required for maintenance of the polar locali-zation of NIP5;1 in the PM.

Polar Localization of NIP5;1 Contributes to Plant Growthunder Low-Boron Conditions

To investigate the physiological significance of the polar lo-calization of NIP5;1 on plant growth, we tested whether themutation affected the boric acid transport activity of NIP5;1. Forthis, we injected cRNAs of wild-type GFP-NIP5;1 and theT18A$T21A$T24A variant into Xenopus laevis oocytes toconduct a boric acid uptake assay (Takano et al., 2006; Mitani-Ueno et al., 2011). We also used cRNA of GFP-tagged NIP1;2,which has a different type of aromatic/arginine selectivity filterfrom NIP5;1 and NIP6;1 and thus is not expected to transportboric acid effectively (Wallace and Roberts, 2005; Mitani et al.,2008; Mitani-Ueno et al., 2011). Identical amounts of cRNAswere injected into the oocytes and similar GFP levels wereobserved in the PM (Figure 6A). The oocytes were incubated inMBS buffer containing 5 mM boric acid for 30 min and theboron concentration was analyzed by inductively coupledplasma-mass spectrometry (ICP-MS). GFP-NIP5;1-expressingoocytes accumulated 2.7- and 1.9-fold higher boron contentsthan water-injected controls and the GFP-NIP1;2-expressingoocytes, respectively (Figure 6B). As expected, the boron contentsof oocytes with wild-type GFP-NIP5;1 and T18A$T21A$T24Adid not differ significantly (Figure 6B), indicating that sub-stitution of the Thr residues did not disturb NIP5;1 borontransport activity.We then investigated the physiological significance of the

polar localization in transgenic Arabidopsis plants. It wasreported previously that the ProNIP5;1:sGFP-NIP5;1(cDNA)

Figure 5. AP2 Is Required for Polar Localization of GFP-NIP5;1 in the PM.

(A) Localization of GFP-NIP5;1 in the epidermal cells of ap2mmutants. The relative fluorescence signal intensities of GFP to FM4-64 are color-coded from0 (black) to 255 (white) with ImageJ software. Bars = 20 mm in longitudinal sections and 50 mm in cross sections.(B)PIs of GFP-NIP5;1 in the epidermal cells of ap2mmutants. Values represent means6 SD. n = a total of 30 to 55 cells from 3 to 6 roots. Asterisks indicatesignificant differences from nip5;1-1 (Student’s t test, P < 0.01).

Polar Localization of NIP5;1 for Boron Transport 833

construct did not fully complement the growth defects of thenip5;1-1 mutant plant under boron limitation, probably due tolow expression levels (Takano et al., 2010; Tanaka et al., 2011).Thus, we tested the ProNIP5;1:GFP-NIP5;1(genomic) con-struct containing a 59-UTR and the genomic sequence ofNIP5;1 from the start codon to the end of the 39-UTR and itsT18A$T21A$T24A variant introduced into a nip5;1-1 back-ground. We measured the mRNA levels and protein levelsbased on GFP signal intensities in T3 homozygous lines withsingle insertions (Figures 7A and 7B). For growth analysis, weselected three linesofGFP-NIP5;1(genomic)wild-type (#3,6,and7)and two lines of GFP-NIP5;1(genomic) T18A$T21A$T24A (#5 and10) with similar mRNA and GFP levels. We also selected two lineswith higher expression of GFP-NIP5;1 T18A$T21A$T24A (#1 and12). These lines showed expression and localization patterns ofwild-typeGFP-NIP5;1andT18A$T21A$T24A inepidermal cells andmature endodermal cells, similar to the patterns in the lines con-taining the cDNAconstruct (Supplemental Figure 9; comparedwithFigures 1A, 2D, and 2F). Using these lines, we characterizedcomplementation of growth of the nip5;1-1mutant by GFP-NIP5;1variants under low-boron conditions (Figures 7C and 7D;Supplemental Figure10).Whenboron in themediumwas increasedfrom 0.03 to 3 mM, Col-0 plants showed improved root and shootgrowth. Thenip5;1-1plants also showedgrowth improvementwithincreasedboron supply, whereas thegrowthwas retarded severelyversus Col-0 even at 3 mM boron, as reported previously (Takanoet al., 2006). Under these conditions, transgenic nip5;1-1 linesexpressing wild-type GFP-NIP5;1 developed similar or slightlylonger primary roots and similar or slightly heavier shoot weights,relative to Col-0, indicating full complementation (Figures 7C and7D; Supplemental Figure 10). By contrast, the GFP-NIP5;1T18A$T21A$T24A variant did not fully complement the growthdefects of the nip5;1-1mutant. At 0.03 mM boron, the primary rootlengths and shoot fresh weights ofGFP-NIP5;1 T18A$T21A$T24Alines were 73 to 87% and only 38 to 47%, respectively, relative to

Col-0 (Figures 7C and 7D). At 3 mMboron, the primary root lengthsand shoot fresh weights of GFP-NIP5;1 T18A$T21A$T24A linesreached levels similar to those of Col-0, although lines #1, 5, and12 still showed significantly lower values (P < 0.01, Student’s t test;Figures7Cand7D). The lineswithhigher expressionofGFP-NIP5;1T18A$T21A$T24A (#1and12) didnotgrowbetter than the lineswithmoderate expression (#5 and 10). In summary, the wild-typeGFP-NIP5;1 protein was able to complement fully the growth de-fects of the nip5;1-1 mutant, whereas the nonphosphorylatableT18A$T21A$T24A variant was not.

Polar Localization of NIP5;1 Is Required for Effective BoronTransport under Low-Boron Conditions

To examine the physiological significance of the polar locali-zation on boron transport, a tracer experiment was conductedusing transgenic plants with similar expression levels of wild-type GFP-NIP5;1 (#3 and 6) and the T18A$T21A$T24A variant(#5 and 10). Plants grown hydroponically with 100 mM 11B-enriched boric acid were divided into two pools. One was in-cubatedwith 1mM10B-enrichedboric acid for 24 h, and theotherwas harvested as a control. The 10B concentration in eachsamplewasdeterminedby ICP-MS, and 10B transported into theshoot during the 24-h periodwas calculated as a function of rootweight. The amount of 10B transported to the shoot in Col-0 dur-ing the 24-h period was about 3 times higher than that in thenip5;1-1 mutant (Figure 7E) and the amount in the transgenicnip5;1-1 lines with wild-type GFP-NIP5;1 was 5 to 29% higherthan in Col-0. This was likely caused by higher expression ofGFP-NIP5;1mRNA, relative to endogenous NIP5;1 (Figure 7A).In the transgenic lines with GFP-NIP5;1 T18A$T21A$T24A, 10Btransport was 27 to 30% lower than in the Col-0. Together,these results indicate that the polar localization of NIP5;1supported the efficient transport of boron in roots under low-boron conditions.

Figure 6. Threonine Residues in the TPG Repeat Are Not Required for Boric Acid Transport Activity of NIP5;1.

(A) and (B)Boric acid transport assay of GFP-taggedNIP1;2, NIP5;1, and theNIP5;1 T18A$T21A$T24A variant in X. laevis oocytes. cRNAs of transcripts ofinterest (;50 ng) were injected into the oocytes. An equivalent volume of water was injected as a negative control.(A) Confocal fluorescence imaging of injected oocytes. Bar = 0.5 mm.(B)Measurement of boron concentration. The oocyteswere incubatedwith 5mMboric acid for 30min and the boron concentrationwasmeasured by ICP-MS. Values represent means 6 SD (n = 4 pools of 5 oocytes pool).

834 The Plant Cell

Figure7. Wild-TypeGFP-NIP5;1 and theT18A$T21A$T24AVariantComplement theGrowthandBoronTransportDefects of thenip5;1-1Mutant inBoron-Limited Conditions.

Polar Localization of NIP5;1 for Boron Transport 835

DISCUSSION

Phosphorylation of Threonine Residues in the ConservedTPG Repeat Mediates the Polar Localization of BoricAcid Channels

In this study, we investigated the localization of members of theNIP family in Arabidopsis root epidermal cells. NIP5;1 and itsclosest homolog NIP6;1 showed strong polar localization, pref-erentially accumulating on the soil side of these cells, and NIP1;2showed a similar, but weak, polar localization (Figure 1A). We alsofoundweakpolar localizationofPIP2;1,NPSN12, andLti6asimilarto thecaseofNIP1;2 (Figure1A;Supplemental Figures2Band2C).A recent report also pointed out the weak polar localization ofPIP2, NPSN12, and a brassinosteroid receptor, BRI1, toward thesoil side in root epidermal cells (Łangowski et al., 2016). Wesuggest that an uneven distribution of certain lipid componentsmightaffect thestability and/or traffickingofmembraneproteins inthe PM of epidermal cells.

The weak polar localization of NIP1;2 helped to identify theamino acid region required for the strong polar localization ofNIP5;1. Replacement of the N-terminal, C-terminal, and centralregions of NIP5;1 with the corresponding regions of NIP1;2demonstrated the requirement for the N-terminal region of NIP5;1for proper polar localization (Figure 1B). We also showed that theN-terminal region of NIP5;1 confers soil-side polar localization onNIP1;2 and PIP2;1 (Figure 1B). Thus, the N-terminal region ofNIP5;1 can be used as a tool in biotechnology to confer polarlocalization, at least on aquaporin homologs. Further trials withdifferent proteins and different NIP5;1 fragments should broadenthis potential and lead to generation of plants with more efficientuse of nutrients and better stress tolerance.

The TPG repeat is conserved in the N-terminal regions ofNIP5;1, NIP6;1 and their rice and maize orthologs (SupplementalFigure 3B). Immunoblot analysis with an antibody recognizingphosphorylated NIP5;1 confirmed that NIP5;1 was phosphory-lated at the Thr residue(s) in the TPG repeat (Figure 2C). Aspsubstitutions of the Thr residues of NIP5;1 resulted in polar lo-calization in both epidermal and endodermal cells, but Ala sub-stitutions compromised this polar localization (Figures 2D to 2F).Ala substitutions of the conserved Thr residues in NIP6;1 alsoaffected polar localization (Supplemental Figure 5). These results

establish an important role of the Thr phosphorylation in theconserved TPG repeat in the polar localization of boric acidchannels. Future studies will examine whether the phosphoryla-tion levels of theThr residues regulate the localization of boric acidchannels in different tissues.

Both Endocytosis and Recycling Maintain the PolarLocalization of NIP5;1

Previous studies have shown the important role of phosphory-lation in regulationof thepolar localizationofPMproteins in animaland plant cells (Offringa andHuang, 2013). In epithelial cells of therenal collecting duct ofmammals, aquaporin 2 (AQP2) functions inwater reabsorption at the apical PM,which faces the lumen. Upondehydration, vasopressin activates cAMP signaling in the ba-solateral PM leading to PKA-mediated phosphorylation ofS256 in the C-terminal tail of AQP2 (Katsura et al., 1997).Phosphorylation of S256 induces translocation of AQP2 frominternal vesicles to the apical PM domain (Fushimi et al., 1997;Katsura et al., 1997; van Balkom et al., 2002). In our study,simultaneous inhibition of recycling by BFA treatment andinhibition of protein synthesis by CHX led to the accumulationof GFP-NIP5;1 in endosomal aggregates. This accumulationwas greatly reduced by the T18A$T21A$T24A substitutions(Figures 4A and 4B). FRAPanalysis showed that the recovery offluorescence in the soil side of the PM did not differ betweenwild-type GFP-NIP5;1 and the T18A$T21A$T24A variant (Fig-ure 3). These results suggest a model whereby Thr phos-phorylation of NIP5;1 enhances endocytosis from the PM,rather than translocation from internal compartments. In mu-tants of clathrin adaptor protein complex 2 (ap2m-1 and ap2m-2),accumulation of GFP-NIP5;1 in BFA-induced endosomal ag-gregates was decreased significantly (Figures 4D and 4E).Moreover, the polar localization of GFP-NIP5;1 was dimin-ished greatly in these lines (Figure 5; Supplemental Figure 8).These results indicated that AP2-dependent endocytosiswas the predominant pathway for the internalization of NIP5;1and is important formaintaining its polar localization. It remainsto be determined whether endocytosis dependent on Thrphosphorylation occurs preferentially from the stele side ofthe PM and whether recycling occurs preferentially to thesoil side. Further studies will attempt to identify the kinase(s)

Figure 7. (continued).

(A)Relative mRNA levels in roots of wild-type and T18A$T21A$T24A lines. ForProNIP5;1:GFP-NIP5;1(genomic) and T18A$T21A$T24A lines, mRNA levelswere determined by RT-mediated real-time PCR analysis. Eleven-day-old plants grown on solid medium supplemented with 100 mMboronwere shifted tosolid medium with 0.3 mM boron for 24 h. Values represent means 6 SD (n = 3 pools of plant samples).(B)Quantificationofprotein levels in root tipsbyfluorescent imaging. Three- to four-day-oldplantsgrownonsolidmediumsupplementedwith100mMboronwere shifted to solid medium with 0.3 mM boron for 24 h. Z-stack images of root tips were taken at 2.5-mm intervals for 22 slices and the sum image wasprojected using ImageJ software (Supplemental Figure 11). Then, the total fluorescence intensity of GFP in root tips in the projected image (ROI: 200 3

600 mm) was obtained with ImageJ. Values represent means 6 SD (n = 3 roots).(C) and (D) Primary root lengths (C) and shoot fresh weight (D) of 1-week-old plants grown with different levels of boron. Values represent means6 SD. n =20 to 28 plants for each line. Asterisks indicate significant differences from Col-0 plants (Student’s t test, P < 0.01).(E) Tracer experiments using stable isotopes of boron. Three-week-old plants grownwith 100 mM11B-enriched (>99%) boric acidwere supplementedwith1mM10B-enriched (>99%)boric acid for 24h. 10Bconcentrations in shoots at times0and24hweremeasuredand theamountof 10B transported into shootswithin 24 h is shown, normalized to root dry weight. Values represent means6 SD (n = 4 plant pools). Different letters indicate significantly different values(one-way ANOVA, LSD-Duncan’s test, P < 0.05; Supplemental Table 1).

836 The Plant Cell

responsible for the Thr phosphorylation and can help to definea possible transcytosis pathway.

Polar Localization of NIP5;1 Facilitates Radial Transportof Boron in Roots and Supports Plant Growth underLow-Boron Conditions

Boron uptake and transport are facilitated by the NIP5;1 boricacid channel and the BOR1 borate exporter in Arabidopsisroots under low-boron conditions (Takano et al., 2008). In thisstudy, we described the expression of NIP5;1 both in matureendodermal cells and in the outermost cell layers of roots(Supplemental Figures 9D, 9E, and 9G). Because of the pres-ence of the apoplastic barrier provided by the Casparian strip,boric acid uptake byNIP5;1 from the soil side and borate exportbyBOR1 from the stele side of the endodermis are important forboron transport into the stele. Substitution of the Thr residueswith Ala affected the polar localization of NIP5;1 in both epi-dermal and endodermal cells (Figures 2D and 2F). In contrastto wild-type GFP-NIP5;1, the GFP-NIP5;1 T18A$T21A$T24Avariant lost its ability to rescue the growth defects of the nip5;1-1mutant (Figures 7C and 7D; Supplemental Figure 10). This isconsistent with the observation that the variant with weakpolar localization transported lower amounts of boron to theshoot than wild-type GFP-NIP5;1 in the nip5;1-1 lines (Figure 7E).These results demonstrate an important physiological rolefor the polar localization of NIP5;1 in boron transport inroots. Importantly, the lines with higher expression of theT18A$T21A$T24A variant (#1 and 12) did not show improvedshoot growth comparedwith the lineswith lower expression (#5and 10; Figures 7A to 7D). This suggests that the amount ofNIP5;1 on the soil side is not the sole factor determining theradial transport efficiency of boron. It seems likely that the influxof boric acid from the stele side of the epidermis and endo-dermis impairs the direction of radial transport of boron bycounteracting the function of BOR1.

Inconclusion,weshowedthe roleofpolar localizationofaborontransport protein both in the efficient translocation of its substrateand in promoting plant growth. Specifically, our results demon-strate that the polar localization of NIP5;1 toward the soil side ofroot epidermal cells is maintained by clathrin-mediated endocy-tosis, is dependent on phosphorylation in the TPG repeat, and isrequired for the effective transport of boron in roots.

METHODS

Plant Materials and Growth Conditions

Arabidopsis thaliana Col-0 was from our laboratory stock. The T-DNAinsertion mutants nip5;1-1 (SALK_122287; Takano et al., 2006), ap2m-1(SALK_08393; Kim et al., 2013), and ap2m-2 (SAIL_165_A05; Yamaokaet al., 2013) were described previously. The nip5;1-1mutant was used fortransformation by the Agrobacterium tumefaciens-mediated in plantamethod (Clough and Bent, 1998). Plasmid vectors described in the nextsection were introduced into Agrobacterium strain GV3101::pMP90. T1plants were selected on half-strength Murashige and Skoog mediumsupplemented with 20 mM hygromycin boron. Then, plants were trans-ferred tomediumwithoutantibioticsandGFPexpressionwasconfirmedbyfluorescence microcopy. T3 homozygous lines with single insertions of

wild-type ProNIP5;1:GFP-NIP5;1(genomic) and the T18A$T21A$T24Avariant were established based on segregation analysis on medium withhygromycin boron. T3 homozygous lines harboring ProNIP5;1(without 59-UTR):GFP-NIP5;1 (Tanaka et al., 2011) were provided by Mayuki Tanaka(The University of Tokyo). ap2m-1 or ap2m-2 homozygous plants ex-pressing GFP-NIP5;1 were selected from F2 populations derived from thecross of ProNIP5;1(without 59-UTR):GFP-NIP5;1/nip5;1 and ap2m-1 orap2m-2 mutants by fluorescence imaging and genotyping using the pri-mers in Supplemental Table 2.

For imaging and growth analyses, seeds were sown on solid medium(Takano et al., 2005), supplemented with 1% (w/v) sucrose, 1.5% (w/v)gellan gum (Wako Pure Chemicals), and various concentrations of boricacid. Purified water was produced using a Milli-Q Advantage A10 puri-fication system (Millipore). Plates were placed vertically in growthchambers at 22°C under a cycle of 18 h light and 6 h dark. For imaging,unless otherwise indicated, plants were supplemented with 10 mM boricacid and grown for 3 to 4 d. For growth analysis, photos of 1-week-oldplants supplemented with 0.03, 0.1, 1.0, or 3.0 mM boric acid were takenand root lengths were measured using the RootNav software (Poundet al., 2013).

Plasmid Construction

Sequences of primers used for plasmid construction are listed inSupplemental Table 2. For the ProNIP5;1:sGFP-NIP5;1(genomic) con-struct, a PCR fragment containing theNIP5;1 genomic sequence from thestart codon to the end of the 39-UTR was fused to a PCR fragment con-taining ProNIP5;1:sGFP that was amplified from ProNIP5;1:sGFP-NIP5;1(Tanaka et al., 2011) using the In-Fusion HD cloning kit (Takara Clontech).To generate the ProNIP5;1:sGFP-NIP5;1(genomic) T18A$T21A$T24Aconstruct, a pair of specific primers containing nucleotide substitutionswas used to perform PCR using the ProNIP5;1:sGFP-NIP5;1(genomic)construct as a template. The 59 and 39 ends of PCR products were fusedusing the In-Fusion HD cloning kit. Similarly, the ProNIP5;1:sGFP-NIP5;1(genomic) T18D$T21D$T24D construct was generated using spe-cific primers containing nucleotide substitutions. The nucleotides alteredto create amino acid substitutions are underlined in Supplemental Table 2.

For high-throughput analysis of the localization of NIPs in the rootepidermal cells, we generated a Gateway destination vector (Invitrogen)containing theNIP5;1 promoter and sGFP. TheGateway vector pGWB506containing the cauliflower mosaic virus 35S (CaMV35S) promoter andsGFP for N-terminal fusion (Nakagawa et al., 2007) was digested withrestriction enzymes HindIII and XbaI (Takara-Bio) to remove the promotersequence. A PCR fragment containing a 2180-bp NIP5;1 promoter se-quence was amplified using Col-0 genomic DNA as a template and wasdigested withHindIII and XbaI. ThisNIP5;1 promoter fragment was ligatedwith the digested pGWB506 to generate the ProNIP5;1:sGFP destinationvector. Tomake entry vectors, the open reading frame (ORF) ofNIP7;1wasamplified from cDNAgenerated fromRNAof Col-0 flowers, and otherNIPsandPIP2;1were amplified from cDNAs generated fromRNAof Col-0 rootsusing the One-Step RT-PCR kit (Qiagen) with specific primers. Then, theORFs of interestwere subcloned into the pENTR/D-TOPOvector by TOPOcloning (Invitrogen). For chimeric constructs, PCR fragments of NIP1;2,NIP5;1, and PIP2;1 and a pENTR/D-TOPO backbone were amplified fromthe entry vectors pENTR/D-TOPO NIP1;2 and pENTR/D-TOPO PIP2;1using specific primers containing overlapping sequences. The amplifiedPCR fragments were then fused using the In-Fusion HD cloning kit. TheGatewayLR reaction (Invitrogen)wasperformedbetween theentry vectors(pENTR/D-TOPObackground) and theProNIP5;1:sGFPdestination vectorto obtain expression constructs.

For the expressionofGFP-NIP5;1ΔNt1–17,ΔNt1–26, T18A$T21A$T24A,T18D$T21D$T24D, T18A, T21A, and T24A variants, constructs were gen-eratedbyPCRusingspecificprimersandthe In-FusionHDcloningkit, based

Polar Localization of NIP5;1 for Boron Transport 837

on the ProNIP5;1:sGFP-NIP5;1 vector (Tanaka et al., 2011). To constructProNIP5;1:sGFP-NIP6;1, the backbone sequence containing ProNIP5;1and sGFP was amplified from the ProNIP5;1:sGFP-NIP5;1 vector andfused with a PCR fragment of NIP6;1 ORF using the In-Fusion HDcloning kit. Similarly, ProNIP5;1:sGFP-NPSN12 and ProNIP5;1:sGFP-LTI6awere generated using theNPSN12 and Lti6a PCR fragments thatwere amplified from GFP-LTI6a- and mCherry-NPSN12-containingplasmids (Geldner et al., 2009; Cutler et al., 2000). Constructs to ex-press the GFP-NIP6;1 T15A$T16A$T19A, T16A, and T19A variantswere generated by PCR using the ProNIP5;1:sGFP-NIP6;1 vector asa template and specific primers containing nucleotide substitutionsand were fused with the In-Fusion HD cloning kit.

The plasmids used for expression in Xenopus laevis oocytes wereconstructed as follows. The ORFs of NIPs N-terminally fused to GFPwere amplified by PCR using specific primers containing BamHI re-striction sites at both 59 ends. The PCR products were digested withBamHI and subcloned into the pXbG-ev1 oocyte expression vector(Preston et al., 1992; Mitani-Ueno et al., 2011) that had been digestedwith BglII. Sequences of PCR fragments, including promoters, codingsequences, and untranslated regions of genes, were confirmed byDNAsequencing.

Quantitative RT-PCR

Plantswere grownon solidmediumcontaining 100mMboron for 11dandshifted to medium containing 0.3 mM boron for 24 h. Total RNA wasextracted using the RNeasy plant mini kit and treated with DNase I(Qiagen). Total RNA (1 mg) was reverse transcribed to cDNA in a 20-mLreaction using the SuperScript III first-strand synthesis system (In-vitrogen) with oligo-d(T)20 primers. Real-time PCR was performed usinga Roche LightCycler 480 real-time System (Roche) and the LightCycler480 SYBR Green I Master kit. The UBQ5 gene was used as an internalcontrol for quantification. The primers used are listed in SupplementalTable 2.

Confocal Microscopy

A Leica TCS-SP5 confocal laser scanning microscope and the TCS-SP8systemwere used for imagingwith the following set of excitation/detectionwavelengths: GFP (488 nm/505–540 nm) and FM4-64 (488 nm/640–750 nm). Plants were grown for 3 to 4 d on vertically placed solid mediumbefore use. Unless otherwise indicated, FM4-64 (Molecular Probes)was used at 4 mM in liquid medium for 30 min prior to imaging to labelmembranes.

To quantify the polarity index in the transverse PM domain in longi-tudinal optical sections (PILongitudinal values), ImageJ software (NIH,http://imagej.nih.gov/ij/)wasused tomeasure themeanvalueofGFPandFM4-64 intensities in the soil- and stele-side halves or the transverse PMdomain (Wakuta et al., 2015; Figure 1C). GFP intensity, normalized byFM4-64 on the soil side, was divided by GFP intensity, normalized byFM4-64 on the stele side. For cross sections, plant rootswere embeddedin medium solidified with 2.5% (w/v) gellan gum (Wako Pure Chemical)and cut with layered razor blades. To quantify the polarity index in thelateral domain in cross sections (PICross), a line 2mmwidewas set to crossthe outer- and innermost portions of epidermal cells (Figure 1D). GFPfluorescence intensity in a 2 3 2-mm region including the PM at theoutermost portion was divided by the GFP fluorescence intensity at theinnermost portion.

Toquantify the rateof intracellular accumulationofGFP-NIP5;1variantsin epidermal cells in the transition zone (six to eight cells after onset ofelongation), we used the polygon tool of ImageJ to measure the meanintensities of the transverse (apical and basal) PM and cytoplasmic region.Then, to define the rate of intracellular accumulation, the intensity of the

cytoplasmic regionwasdividedby the intensityof thesumof the transversePM and cytoplasmic regions (Yoshinari et al., 2016).

FRAP experiments were performed using FRAP Wizard TCS-SP8(Leica), equipped with an argon laser and an HC PL APO CS 340 waterimmersion lens. For imaging before and after photobleaching, a 488-nmdiode laser was used at low levels (<2% transmission) to minimizephotobleaching during measurements. A circular region of interest (ROI)6 mm in diameter was bleached with a 488-nm argon laser at 100%transmission for 3 s. The “Zoom In” option was used in FRAP Wizard.Recovery of fluorescence was recorded at 5-min intervals for up to30 min. The percentage of recovery was calculated based on theequation: (It2 Ip) / (I02 Ip)3 100, where It is theGFP intensity at each timepoint, Ip is the GFP intensity after bleaching, and I0 is the GFP intensitybefore bleaching (Luu et al., 2012).

Immunoprecipitation

Roots of 2-week-old plants (1.5 to 4.5 g) were harvested and homogenizedusing a Multi-Beads Shocker (Yasui Kikai) in buffer (2 mL per 1 g sample,250mMTris, pH 7.5, 100mMNaCl, 5 mMDTT, 0.5mMNa2MoO4, 1.5 mMNaF, 0.5 mMNa3VO4, and 2%Nonidet P-40) supplemented with proteaseinhibitors (cOmplete Mini, EDTA-free [Roche] and 0.5 mg L21 Pefabloc SC[Roche]) andPhosSTOPphosphatase inhibitor cocktail (Roche) on ice. Thelysate was centrifuged (9100g, and then 20,000g, 15 min each at 4°C) toremove cellular debris.

Immunoprecipitations (IPs) were performed using the mMACS GFP-tagged protein isolation kit (Miltenyi Biotec) following the supplier’s pro-tocol with the following modification. After rinsing the columns once with100mLofwashbuffer 2 (20mMTris-HCl, pH7.5), the IP sampleswith 30mLof microbeads were collected in;50 mL of NE buffer (50 mM HEPES, pH7.5, 100 mM NaCl, 2 mM DTT, and 0.01% Brij 35).

Generation of an Anti-Phospho-Threonine Antibody

The anti-phospho-Thr antibody of NIP5;1 was generated by SCRUM asfollows. A phosphorylated peptide [Cys-MAPP(pT)PG(pT)PG(pT)PG](>80% purity) was synthesized and used to immunize rabbits. At 6 weeksafter immunization, 20 mL of serum was collected and affinity purifiedusing the phosphorylated peptide. To remove antibodies recognizingnonphosphorylated sequences, the eluate was absorbed with a non-phosphorylated peptide (Cys-MAPPTPGTPGTPG) on an affinity col-umn. The flow-through fraction contained the anti-phospho-Thr NIP5;1antibodies.

Analysis of Phosphorylation by Immunoblotting

For phosphatase treatment, 0.5 mL of lambda protein phosphatase(New England Biolabs) and 1 mL of 10 mMMnCl2 were added to tubescontaining 8.5 mL of IP sample including microbeads and were thenincubated at 30°C for 1 h. Control samples were supplemented with anequivalent volume of NE buffer and MnCl2. Then, 4 mL of 43 LDSsample buffer (Thermo Fischer Scientific) and DTT (166 mM finalconcentration) were added and the samples were denatured at 85°Cfor 10 min.

For immunoblotting, sampleswere separated on aNuPAGENovex 4 to12% Bis-Tris gel (Thermo Fischer Scientific) and transferred to a PVDFmembrane (Merck Millipore) by a standard wet-transfer method. Themembrane was immediately blocked with 3% (w/v) skim milk (Difco) inTBST (50 mM Tris, 150 mM NaCl, and 0.1% Tween 20) for 1 h at roomtemperature. The membrane was then incubated with anti-GFP mono-clonal antibody (1:2000; mouse IgG1-k; Nacalai Tesque) or the anti-phosphoTNIP5;1 polyclonal antibody (1:500) diluted with 3% (w/v) skimmilk in TBST for 1 to 2 h. The secondary antibody was either horseradish

838 The Plant Cell

peroxidase (HRP)-conjugated anti-mouse IgG antibody (1:10,000; GEHealthcare; cat. no. NV931) or HRP-conjugated anti-rabbit IgG antibody(1:10,000; GE Healthcare; cat. no. NV934). After washing the membranewith TBST, 2 mL of Luminata Forte Western HRP substrate (Merck Milli-pore) was applied and luminescence was imaged with a LuminoGraph Isystem (ATTO).

Boron Uptake Assay in X. laevis Oocytes

Capped cRNA was transcribed in vitro from XbaI-linearized vectorsusing themMESSAGEmMACHINE kit (Ambion). Oocytes injectedwiththe cRNA of interest using an injector (Drummond Nanoject II Auto-Nanoliter) were kept inMBSbuffer [88.0mMNaCl, 1.0mMKCl, 2.4mMNaHCO3, 15.0 mMTris-HCl, pH 7.6, 0.3 mMCa(NO3)2, 0.41mMCaCl2,0.82 mM MgSO4, 10 mg mL21 sodium penicillin, and 10 mg mL21

streptomycin sulfate] for 48 h at 18°C. Then, oocytes with GFP signalswere selected under a fluorescence microscope (MVX10; Olympus)and incubated in medium supplemented with 5 mM boric acid for30 min. Five oocytes were pooled in DigiTUBEs (GL Sciences) andwashed with 1 mL of boron-free MBS buffer five times. Oocytes wereincubated in 500 mL of concentrated HNO3 for 20 min, followed byincubation for 20 min in a 100°C prewarmed DigiPREP MS apparatus(GL Sciences). The tubes were further incubated at 110°C and 120°Cfor 20 min each for digestion. Finally, 99.0 mL of concentrated HNO3

was added to dissolve the samples and 2900 mL of Milli-Q water wasadded. Boron contents in oocytes were determined by ICP-MS asdescribed below.

Tracer Experiment Using Stable Isotopes of Boron

Two-week-old plants grown on solid medium containing 100 mM 11B-enriched (>99%)boric acidwereshifted tohydroponicmediawith100mM11B-enriched boric acid for 1week. For each line, 12 plants were grown inone pot; half of the plants were sampled at time 0 and the others weresupplemented with 1 mM 10B-enriched (>99%) boric acid for 24 h. Theroot and shoot tissueswere dried at 60°C for 3 d and the dryweightsweremeasured. The samples were digested for 24 h in DigiTUBEs (GL Sci-ences) with 6 mL of concentrated nitric acid (for boron determination;Wako Pure Chemical) at 110°C in a DigiPREP MS apparatus (GL Sci-ences). After the nitric acid solution was evaporated, 10 mL of 2%HNO3

was added to dissolve the samples. The solution was filtered witha DigiFILTER (0.45mm; GL Sciences) to remove solid materials and usedfor ICP-MS.

Boron Determination by ICP-MS

Boron concentrations were determined using an ICP-MS ELAN DRC-eequippedwith ESI Autosamplers (Perkin-Elmer). 10Bwasmeasured for thetracer experiment and 11B was measured for the boron concentration inoocytes. Purified water was produced using the Milli-Q Advantage A10purification system (Millipore).

Statistical Analyses

Dataweresubjected toanalysis of variance using theSPSSsoftware (IBM).Statistical significance was assessed by least significant differences andDuncan’s multiple range tests.

Accession Numbers

The ArabidopsisGenome Initiative locus identifiers for the genesmentionedin this article are as follows:NIP1;1 (At4g19030),NIP1;2 (At4g18910),NIP2;1(At2g34390), NIP3;1 (At1g31885),NIP4;1 (At5g37810), NIP5;1 (At4g10380),

NIP6;1 (At1g80760), NIP7;1 (At3g06100), PIP2;1 (At3g53420), NPSN12(At1g48240), LTI6a (At3g05880), and AP2M (At5g46630). T-DNA insertionmutantsareas follows:nip5;1-1 (SALK_122287;Takanoetal.,2006),ap2m-1(SALK_08393;Kimetal., 2013),andap2m-2 (SAIL_165_A05;Yamaokaetal.,2013).

Supplemental Data

Supplemental Figure 1. Distinct Localization of NIPs in RootEpidermal Cells.

Supplemental Figure 2. Weakly Polar Localization of NPSN12 andPIP2;1 in Root Epidermal Cells.

Supplemental Figure 3. Alignment of NIP Homolog Sequences.

Supplemental Figure 4. Deletion of the N-Terminal Sequence InducesVacuolar Trafficking of NIP5;1.

Supplemental Figure 5. Conserved Threonine Residues in theN-Terminal Region Are Essential for Polar Localization of NIP6;1.

Supplemental Figure 6. Time-Course Imaging of Newly-SynthesizedGFP-NIP5;1.

Supplemental Figure 7. CHX Inhibits the Appearance of GFP-NIP5;1.

Supplemental Figure 8. AP2M Is Required for Polar Localization ofGFP-NIP5;1 in the PM.

Supplemental Figure 9. Polar Localization of GFP-NIP5;1 Wild Typeand Weakly Polar Localization of the GFP-NIP5;1 T18A$T21A$T24AVariant in ProNIP5;1:GFP-NIP5;1(genomic)/nip5;1-1 Plants.

Supplemental Figure 10. Growth Characterization of nip5;1-1 Lineswith Wild-Type GFP-NIP5;1 and the T18A$T21A$T24A Variant underBoron-Limited Conditions.

Supplemental Figure 11. Quantification of GFP-NIP5;1 Protein Levelsin Root Tips by Fluorescent Imaging.

Supplemental Table 1. One-Way ANOVA Data.

Supplemental Table 2. Primers Used in This Study

ACKNOWLEDGMENTS

We thank Kayo Konishi and Tomoko Shimizu for technical assistance;Toshihiro Watanabe, Toshiaki Ito (Hokkaido University), Naoki Yamaji(Okayama University), and Koji Kasai (The University of Tokyo) for valu-able comments and technical guidance; and Niko Geldner (University ofLausanne), Toru Fujiwara, and Mayuki Tanaka (The University of Tokyo)for valuable comments and materials. We also thank Gabriel Schaaf(Tübingen University) for critical reading of the manuscript; and ShigekiTakada, Yuko Imai, Hiroaki Koyama (Hokkaido University), TsuyoshiNakagawa (Shimane University), Inhwan Hwang (Pohang University ofScience and Technology), and the ABRC for materials. This work wassupported by the NEXT program (GS001) and the Grant-in-Aid for YoungScientists (A) (26712007) from the Japan Society for the Promotion ofScience, theYoung InvestigatorsGrant from theHuman Frontier ScienceProgram (RGY0090/2011), and a research grant from the Naito Foun-dation to J.T.

AUTHOR CONTRIBUTIONS

S.W., S.N., and J.T. designed the research. S.W. performed the experi-ments and analyzed the data. A.Y. generated GFP-NIP5;1 ap2m lines andperformed imaging analysis using them. T.S. and I.H.-N. generated and

Polar Localization of NIP5;1 for Boron Transport 839

characterized ap2m mutants. N.M.-U. and J.F.M. performed and sup-ported setup of boron uptake assay in oocytes. S.W. and J.T. wrote thearticlewith improvements fromA.Y., T.S., I.H.-N.,N.M.-U., J.F.M., andS.N.

Received November 2, 2016; revised January 26, 2017; accepted March23, 2017; published March 24, 2017.

REFERENCES

Alassimone, J., Naseer, S., and Geldner, N. (2010). A developmentalframework for endodermal differentiation and polarity. Proc. Natl.Acad. Sci. USA 107: 5214–5219.

Barberon, M., Dubeaux, G., Kolb, C., Isono, E., Zelazny, E., andVert, G. (2014). Polarization of IRON-REGULATED TRANSPORTER1 (IRT1) to the plant-soil interface plays crucial role in metal ho-meostasis. Proc. Natl. Acad. Sci. USA 111: 8293–8298.

Barberon, M., and Geldner, N. (2014). Radial transport of nutrients: theplant root as a polarized epithelium. Plant Physiol. 166: 528–537.

Bashline, L., Li, S., Anderson, C.T., Lei, L., and Gu, Y. (2013). The en-docytosis of cellulose synthase in Arabidopsis is dependent on m2,a clathrin-mediated endocytosis adaptin. Plant Physiol. 163: 150–160.

Bienert, G.P., Thorsen, M., Schüssler, M.D., Nilsson, H.R., Wagner,A., Tamás, M.J., and Jahn, T.P. (2008). A subgroup of plantaquaporins facilitate the bi-directional diffusion of As(OH)3 andSb(OH)3 across membranes. BMC Biol. 6: 26.

Choi, W.G., and Roberts, D.M. (2007). Arabidopsis NIP2;1, a majorintrinsic protein transporter of lactic acid induced by anoxic stress.J. Biol. Chem. 282: 24209–24218.

Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana.Plant J. 16: 735–743.

Cutler, S.R., Ehrhardt, D.W., Griffitts, J.S., and Somerville, C.R.(2000). Random GFP:cDNA fusions enable visualization of sub-cellular structures in cells of Arabidopsis at a high frequency. Proc.Natl. Acad. Sci. USA 97: 3718–3723.

Dordas, C., Chrispeels, M.J., and Brown, P.H. (2000). Permeabilityand channel-mediated transport of boric acid across membranevesicles isolated from squash roots. Plant Physiol. 124: 1349–1362.

Doyle, S.M., Haeger, A., Vain, T., Rigal, A., Viotti, C., Łangowska,M., Ma, Q., Friml, J., Raikhel, N.V., Hicks, G.R., and Robert, S.(2015). An early secretory pathway mediated by GNOM-LIKE 1 andGNOM is essential for basal polarity establishment in Arabidopsisthaliana. Proc. Natl. Acad. Sci. USA 112: E806–E815.

Durbak, A.R., Phillips, K.A., Pike, S., O’Neill, M.A., Mares, J.,Gallavotti, A., Malcomber, S.T., Gassmann, W., and McSteen,P. (2014). Transport of boron by the tassel-less1 aquaporin is criticalfor vegetative and reproductive development in maize. Plant Cell26: 2978–2995.

Fan, L., Hao, H., Xue, Y., Zhang, L., Song, K., Ding, Z., Botella,M.A., Wang, H., and Lin, J. (2013). Dynamic analysis of ArabidopsisAP2 s subunit reveals a key role in clathrin-mediated endocytosisand plant development. Development 140: 3826–3837.

Fan, L., Li, R., Pan, J., Ding, Z., and Lin, J. (2015). Endocytosis andits regulation in plants. Trends Plant Sci. 20: 388–397.

Feraru, E., Feraru, M.I., Kleine-Vehn, J., Martinière, A., Mouille, G.,Vanneste, S., Vernhettes, S., Runions, J., and Friml, J. (2011). PINpolarity maintenance by the cell wall in Arabidopsis. Curr. Biol. 21:338–343.

Friml, J., et al. (2004). A PINOID-dependent binary switch in apical-basal PIN polar targeting directs auxin efflux. Science 306:862–865.

Funakawa, H., and Miwa, K. (2015). Synthesis of borate cross-linkedrhamnogalacturonan II. Front. Plant Sci. 6: 223.

Fushimi, K., Sasaki, S., and Marumo, F. (1997). Phosphorylation ofserine 256 is required for cAMP-dependent regulatory exocytosisof the aquaporin-2 water channel. J. Biol. Chem. 272: 14800–14804.

Ganguly, A., Sasayama, D., and Cho, H.T. (2012). Regulation of thepolarity of protein trafficking by phosphorylation. Mol. Cells 33:423–430.

Geldner, N., Anders, N., Wolters, H., Keicher, J., Kornberger, W.,Muller, P., Delbarre, A., Ueda, T., Nakano, A., and Jürgens, G.(2003). The Arabidopsis GNOM ARF-GEF mediates endosomal re-cycling, auxin transport, and auxin-dependent plant growth. Cell112: 219–230.

Geldner, N., Dénervaud-Tendon, V., Hyman, D.L., Mayer, U.,Stierhof, Y.-D., and Chory, J. (2009). Rapid, combinatorial analy-sis of membrane compartments in intact plants with a multicolormarker set. Plant J. 59: 169–178.

Gordon-Weeks, R., Tong, Y., Davies, T.G.E., and Leggewie, G.(2003). Restricted spatial expression of a high-affinity phosphatetransporter in potato roots. J. Cell Sci. 116: 3135–3144.

Hanaoka, H., Uraguchi, S., Takano, J., Tanaka, M., and Fujiwara, T.(2014). OsNIP3;1, a rice boric acid channel, regulates boron distri-bution and is essential for growth under boron-deficient conditions.Plant J. 78: 890–902.

Ishii, T., and Matsunaga, T. (1996). Isolation and characterization ofa boron-rhamnogalacturonan-II complex from cell walls of sugarbeet pulp. Carbohydr. Res. 284: 1–9.

Kania, U., Fendrych, M., and Friml, J. (2014). Polar delivery in plants;commonalities and differences to animal epithelial cells. Open Biol.4: 140017.

Katsura, T., Gustafson, C.E., Ausiello, D.A., and Brown, D. (1997).Protein kinase A phosphorylation is involved in regulated exocytosisof aquaporin-2 in transfected LLC-PK1 cells. Am. J. Physiol. 272:F817–F822.

Kiba, T., Feria-Bourrellier, A.-B., Lafouge, F., Lezhneva, L., Boutet-Mercey, S., Orsel, M., Bréhaut, V., Miller, A., Daniel-Vedele, F.,Sakakibara, H., and Krapp, A. (2012). The Arabidopsis nitratetransporter NRT2.4 plays a double role in roots and shoots of ni-trogen-starved plants. Plant Cell 24: 245–258.

Kim, S.Y., Xu, Z.-Y.Z.-Y., Song, K., Kim, D.H., Kang, H., Reichardt,I., Sohn, E.J.J., Friml, J., Juergens, G., and Hwang, I. (2013).Adaptor protein complex 2-mediated endocytosis is crucial for malereproductive organ development in Arabidopsis. Plant Cell 25:2970–2985.

Kleine-Vehn, J., et al. (2011). Recycling, clustering, and endocytosisjointly maintain PIN auxin carrier polarity at the plasma membrane.Mol. Syst. Biol. 7: 540.

Kleine-Vehn, J., Dhonukshe, P., Sauer, M., Brewer, P.B.,Wisniewska, J., Paciorek, T., Benková, E., and Friml, J. (2008).ARF GEF-dependent transcytosis and polar delivery of PIN auxincarriers in Arabidopsis. Curr. Biol. 18: 526–531.

Kleine-Vehn, J., Huang, F., Naramoto, S., Zhang, J., Michniewicz,M., Offringa, R., and Friml, J. (2009). PIN auxin efflux carrier po-larity is regulated by PINOID kinase-mediated recruitment intoGNOM-independent trafficking in Arabidopsis. Plant Cell 21: 3839–3849.

Kobayashi, M., Matoh, T., and Azuma, J. (1996). Two chains ofrhamnogalacturonan II are cross-linked by borate-diol ester bondsin higher plant cell walls. Plant Physiol. 110: 1017–1020.

Łangowski, L., Ruzicka, K., Naramoto, S., Kleine-Vehn, J., andFriml, J. (2010). Trafficking to the outer polar domain defines theroot-soil interface. Curr. Biol. 20: 904–908.

840 The Plant Cell

Łangowski, Ł., Wabnik, K., Li, H., Vanneste, S., Naramoto, S.,Tanaka, H., and Friml, J. (2016). Cellular mechanisms for cargodelivery and polarity maintenance at different polar domains in plantcells. Cell Discov. 2: 16018.

Leonard, A., et al. (2014). tassel-less1 encodes a boron channelprotein required for inflorescence development in maize. Plant CellPhysiol. 55: 1044–1054.

Li, T., Choi, W.-G., Wallace, I.S., Baudry, J., and Roberts, D.M.(2011). Arabidopsis thaliana NIP7;1: an anther-specific boric acidtransporter of the aquaporin superfamily regulated by an unusualtyrosine in helix 2 of the transport pore. Biochemistry 50: 6633–6641.

Löfke, C., Luschnig, C., and Kleine-Vehn, J. (2013). Post-translational modification and trafficking of PIN auxin efflux carriers.Mech. Dev. 130: 82–94.

Luu, D.-T.T., Martinière, A., Sorieul, M., Runions, J., and Maurel, C.(2012). Fluorescence recovery after photobleaching reveals highcycling dynamics of plasma membrane aquaporins in Arabidopsisroots under salt stress. Plant J. 69: 894–905.

Ma, J.F., Tamai, K., Yamaji, N., Mitani, N., Konishi, S., Katsuhara,M., Ishiguro, M., Murata, Y., and Yano, M. (2006). A silicontransporter in rice. Nature 440: 688–691.

Ma, J.F., Yamaji, N., Mitani, N., Tamai, K., Konishi, S., Fujiwara, T.,Katsuhara, M., and Yano, M. (2007). An efflux transporter of siliconin rice. Nature 448: 209–212.

Malínská, K., Jelínková, A., and Petrá�sek, J. (2014). The use of FMdyes to analyze plant endocytosis. Methods Mol. Biol. 1209: 1–11.

Marschner, P. (2012). Marschner’s Mineral Nutrition of Higher Plants,(San Diego, CA: Academic Press).

Maurel, C., Boursiac, Y., Luu, D.-T., Santoni, V., Shahzad, Z., andVerdoucq, L. (2015). Aquaporins in plants. Physiol. Rev. 95: 1321–1358.

Mitani-Ueno, N., Yamaji, N., Zhao, F.J., and Ma, J.F. (2011). Thearomatic/arginine selectivity filter of NIP aquaporins plays a criticalrole in substrate selectivity for silicon, boron, and arsenic. J. Exp.Bot. 62: 4391–4398.

Mitani, N., Yamaji, N., and Ma, J.F. (2008). Characterization ofsubstrate specificity of a rice silicon transporter, Lsi1. Pflugers Arch.456: 679–686.

Miwa, K., Wakuta, S., Takada, S., Ide, K., Takano, J., Naito, S.,Omori, H., Matsunaga, T., and Fujiwara, T. (2013). Roles of BOR2,a boron exporter, in cross linking of rhamnogalacturonan II and rootelongation under boron limitation in Arabidopsis. Plant Physiol. 163:1699–1709.

Mizutani, M., Watanabe, S., Nakagawa, T., and Maeshima, M.(2006). Aquaporin NIP2;1 is mainly localized to the ER membraneand shows root-specific accumulation in Arabidopsis thaliana. PlantCell Physiol. 47: 1420–1426.

Nagarajan, Y., et al. (2015). A barley efflux transporter operates ina Na+-dependent manner, as revealed by a multidisciplinary plat-form. Plant Cell 28: 202–218.

Nakagawa, T., et al. (2007). Improved Gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenicanalysis of plants. Biosci. Biotechnol. Biochem. 71: 2095–2100.

Naramoto, S., Otegui, M.S., Kutsuna, N., de Rycke, R., Dainobu, T.,Karampelias, M., Fujimoto, M., Feraru, E., Miki, D., Fukuda, H.,Nakano, A., and Friml, J. (2014). Insights into the localization andfunction of the membrane trafficking regulator GNOM ARF-GEF atthe Golgi apparatus in Arabidopsis. Plant Cell 26: 3062–3076.

Noguchi, K., Yasumori, M., Imai, T., Naito, S., Matsunaga, T., Oda,H., Hayashi, H., Chino, M., and Fujiwara, T. (1997). bor1-1, anArabidopsis thalianamutant that requires a high level of boron. PlantPhysiol. 115: 901–906.

Offringa, R., and Huang, F. (2013). Phosphorylation-dependenttrafficking of plasma membrane proteins in animal and plant cells.J. Integr. Plant Biol. 55: 789–808.

O’Neill, M.A., Warrenfeltz, D., Kates, K., Pellerin, P., Doco, T.,Darvill, A.G., and Albersheim, P. (1996). Rhamnogalacturonan-II,a pectic polysaccharide in the walls of growing plant cell, formsa dimer that is covalently cross-linked by a borate ester. In vitroconditions for the formation and hydrolysis of the dimer. J. Biol.Chem. 271: 22923–22930.

Paez Valencia, J., Goodman, K., and Otegui, M.S. (2016). Endocy-tosis and endosomal trafficking in plants. Annu. Rev. Plant Biol. 67:309–335.

Pound, M.P., French, A.P., Atkinson, J.A., Wells, D.M., Bennett,M.J., and Pridmore, T. (2013). RootNav: navigating images ofcomplex root architectures. Plant Physiol. 162: 1802–1814.

Preston, G.M., Carroll, T.P., Guggino, W.B., and Agre, P. (1992).Appearance of water channels in Xenopus oocytes expressing redcell CHIP28 protein. Science 256: 385–387.

Ruzicka, K., et al. (2010). Arabidopsis PIS1 encodes the ABCG37transporter of auxinic compounds including the auxin precursorindole-3-butyric acid. Proc. Natl. Acad. Sci. USA 107: 10749–10753.

Sakurai, G., Satake, A., Yamaji, N., Mitani-Ueno, N., Yokozawa, M.,Feugier, F.G., and Ma, J.F. (2015). In silico simulation modelingreveals the importance of the Casparian strip for efficient siliconuptake in rice roots. Plant Cell Physiol. 56: 631–639.

Sasaki, A., Yamaji, N., Yokosho, K., and Ma, J.F. (2012). Nramp5 isa major transporter responsible for manganese and cadmium up-take in rice. Plant Cell 24: 2155–2167.

Shorrocks, V.M. (1997). The occurrence and correction of borondeficiency. Plant Soil 193: 121–148.

Strader, L.C., and Bartel, B. (2009). The Arabidopsis PLEIOTROPICDRUG RESISTANCE8/ABCG36 ATP binding cassette transportermodulates sensitivity to the auxin precursor indole-3-butyric acid.Plant Cell 21: 1992–2007.

Takano, J., Miwa, K., and Fujiwara, T. (2008). Boron transportmechanisms: collaboration of channels and transporters. TrendsPlant Sci. 13: 451–457.

Takano, J., Miwa, K., Yuan, L., von Wirén, N., and Fujiwara, T.(2005). Endocytosis and degradation of BOR1, a boron transporterof Arabidopsis thaliana, regulated by boron availability. Proc. Natl.Acad. Sci. USA 102: 12276–12281.

Takano, J., Noguchi, K., Yasumori, M., Kobayashi, M., Gajdos, Z.,Miwa, K., Hayashi, H., Yoneyama, T., and Fujiwara, T. (2002).Arabidopsis boron transporter for xylem loading. Nature 420: 337–340.

Takano, J., Tanaka, M., Toyoda, A., Miwa, K., Kasai, K., Fuji, K.,Onouchi, H., Naito, S., and Fujiwara, T. (2010). Polar localizationand degradation of Arabidopsis boron transporters through distincttrafficking pathways. Proc. Natl. Acad. Sci. USA 107: 5220–5225.

Takano, J., Wada, M., Ludewig, U., Schaaf, G., von Wirén, N., andFujiwara, T. (2006). The Arabidopsis major intrinsic protein NIP5;1is essential for efficient boron uptake and plant development underboron limitation. Plant Cell 18: 1498–1509.

Takano, J., Yamagami, M., Noguchi, K., Hayashi, H., and Fujiwara,T. (2001). Preferential translocation of boron to young leaves inArabidopsis thaliana regulated by the BOR1 gene. Soil Sci. PlantNutr. 47: 345–357.

Tamura, K., Shimada, T., Ono, E., Tanaka, Y., Nagatani, A.,Higashi, S.I., Watanabe, M., Nishimura, M., and Hara-Nishimura, I. (2003). Why green fluorescent fusion proteins havenot been observed in the vacuoles of higher plants. Plant J. 35: 545–555.

Polar Localization of NIP5;1 for Boron Transport 841

Tanaka, M., Sotta, N., Yamazumi, Y., Yamashita, Y., Miwa, K.,Murota, K., Chiba, Y., Hirai, M.Y., Akiyama, T., Onouchi, H.,Naito, S., and Fujiwara, T. (2016). The minimum open readingframe, AUG-stop, induces boron-dependent ribosome stalling andmRNA degradation. Plant Cell 28: 2830–2849.

Tanaka, M., Takano, J., Chiba, Y., Lombardo, F., Ogasawara, Y.,Onouchi, H., Naito, S., and Fujiwara, T. (2011). Boron-dependentdegradation of NIP5;1 mRNA for acclimation to excess boronconditions in Arabidopsis. Plant Cell 23: 3547–3559.

Tanaka, M., Wallace, I.S., Takano, J., Roberts, D.M., and Fujiwara,T. (2008). NIP6;1 is a boric acid channel for preferential transport ofboron to growing shoot tissues in Arabidopsis. Plant Cell 20: 2860–2875.

Uehara, M., Wang, S., Kamiya, T., Shigenobu, S., Yamaguchi, K.,Fujiwara, T., Naito, S., and Takano, J. (2014). Identification andcharacterization of an Arabidopsis mutant with altered localizationof NIP5;1, a plasma membrane boric acid channel, reveals the re-quirement for D-galactose in endomembrane organization. PlantCell Physiol. 55: 704–714.

Ueno, D., Sasaki, A., Yamaji, N., Miyaji, T., Fujii, Y., Takemoto, Y.,Moriyama, S., Che, J., Moriyama, Y., Iwasaki, K., and Ma, J.F.(2015). A polarly localized transporter for efficient manganese up-take in rice. Nat. Plants 1: 15170.

Ueno, D., Yamaji, N., and Ma, J.F. (2009). Further characterization offerric-phytosiderophore transporters ZmYS1 and HvYS1 in maizeand barley. J. Exp. Bot. 60: 3513–3520.

van Balkom, B.W.M., Savelkoul, P.J.M., Markovich, D., Hofman, E.,Nielsen, S., van der Sluijs, P., and Deen, P.M.T. (2002). The role ofputative phosphorylation sites in the targeting and shuttling of theaquaporin-2 water channel. J. Biol. Chem. 277: 41473–41479.

Wakuta, S., Mineta, K., Amano, T., Toyoda, A., Fujiwara, T., Naito,S., and Takano, J. (2015). Evolutionary divergence of plant borateexporters and critical amino acid residues for the polar localizationand boron-dependent vacuolar sorting of AtBOR1. Plant CellPhysiol. 56: 852–862.

Wallace, I.S., and Roberts, D.M. (2005). Distinct transport selectivity oftwo structural subclasses of the nodulin-like intrinsic protein family ofplant aquaglyceroporin channels. Biochemistry 44: 16826–16834.

Wallace, I.S., Choi, W.-G., and Roberts, D.M. (2006). The structure,function and regulation of the nodulin 26-like intrinsic protein familyof plant aquaglyceroporins. Biochim. Biophys. Acta 1758: 1165–1175.

Yamaoka, S., Shimono, Y., Shirakawa, M., Fukao, Y., Kawase, T.,Hatsugai, N., Tamura, K., Shimada, T., and Hara-Nishimura, I.(2013). Identification and dynamics of Arabidopsis adaptor protein-2 complex and its involvement in floral organ development. PlantCell 25: 2958–2969.

Yoshinari, A., Fujimoto, M., Ueda, T., Inada, N., Naito, S., andTakano, J. (2016). DRP1-dependent endocytosis is essential forpolar localization and boron-induced degradation of the boratetransporter BOR1 in Arabidopsis thaliana. Plant Cell Physiol. 57:1985–2000.

Zulawski, M., Braginets, R., and Schulze, W.X. (2013). PhosPhAtgoes kinases–searchable protein kinase target information in theplant phosphorylation site database PhosPhAt. Nucleic Acids Res.41: D1176–D1184.

Zwiewka, M., Nodzynski, T., Robert, S., Vanneste, S., and Friml, J.(2015). Osmotic stress modulates the balance between exocytosisand clathrin-mediated endocytosis in Arabidopsis thaliana. Mol.Plant 8: 1175–1187.

842 The Plant Cell

DOI 10.1105/tpc.16.00825; originally published online March 24, 2017; 2017;29;824-842Plant Cell

Feng Ma, Satoshi Naito and Junpei TakanoSheliang Wang, Akira Yoshinari, Tomoo Shimada, Ikuko Hara-Nishimura, Namiki Mitani-Ueno, Jian

Boron Transport in Arabidopsis RootsPolar Localization of the NIP5;1 Boric Acid Channel Is Maintained by Endocytosis and Facilitates

 This information is current as of October 24, 2020

 

Supplemental Data

/content/suppl/2019/07/17/tpc.16.00825.DC3.html /content/suppl/2017/05/02/tpc.16.00825.DC2.html /content/suppl/2017/03/24/tpc.16.00825.DC1.html

References /content/29/4/824.full.html#ref-list-1

This article cites 80 articles, 35 of which can be accessed free at:

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists