Preferential Distribution of Boron to DevelopingTissues Is Mediated by the Intrinsic Protein OsNIP31[OPEN]

Ji Feng Shao,a,b Naoki Yamaji,a Xin Wei Liu,a,c Kengo Yokosho,a Ren Fang Shen,b and Jian Feng Maa,b,2

aInstitute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, JapanbState Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy ofSciences, Nanjing 210008, ChinacMicroelement Research Center, Huazhong Agricultural University, Wuhan 430070, China

ORCID IDs: 0000-0003-3935-268X (K.Y.); 0000-0003-3411-827X (J.F.M.).

Boron is especially required for the growth of meristem and reproductive organs, but the molecular mechanisms underlying thepreferential distribution of B to these developing tissues are poorly understood. Here, we show evidence that a member ofnodulin 26-like intrinsic protein (NIP), OsNIP3;1, is involved in this preferential distribution in rice (Oryza sativa). OsNIP3;1 washighly expressed in the nodes and its expression was up-regulated by B deficiency, but down-regulated by high B. OsNIP3;1 waspolarly localized at the xylem parenchyma cells of enlarged vascular bundles of nodes facing toward the xylem vessels.Furthermore, this protein was rapidly degraded within a few hours in response to high B. Knockout of this gene hardlyaffected the uptake and root-to-shoot translocation of B, but altered B distribution in different organs in the above-groundparts, decreased distribution of B to the new leaves, and increased distribution to the old leaves. These results indicate thatOsNIP3;1 located in the nodes is involved in the preferential distribution of B to the developing tissues by unloading B from thexylem in rice and that it is regulated at both transcriptional and protein level in response to external B level.

Boron (B) is an essential micronutrient for plantgrowth and development. Its major physiologicalfunction is to maintain the structure of the cell wall bycross linking pectic polysaccharides through borate-diol bonding of two rhamnogalacturonan II molecules(Matoh 1997; O’Neill et al., 2001). The requirement of Branges from 5 mg/kg to 100 mg/kg, depending onplant species (Marschner 2012). There is a good corre-lation between B requirement and pectin content of thecell wall (Matoh et al., 1993). Except some plant speciesproducing sugar alcohols such as sorbitol and manni-tol, B is immobile in most plant species (Brown andShelp, 1997). Therefore, a continuous supply of B isrequired to maintain growth of newly developing tis-sues, and deficiency of B will result in the cessation ofroot elongation, reduced leaf expansion, and loss of

fertility (Dell and Huang 1997; Loomis and Durst 1992;Marschner 1995). On the other hand, B also showstoxicity to plants when present in excess, whichsymptoms are characterized by chlorosis and necrosisat the tips of older leaves (Schnurbusch et al., 2010).Both B deficiency and toxicity cause crop losses inmanyareas of the world (Nable et al., 1997; Shorrocks, 1997).

Plant roots take up B in the form of boric acid. Twodifferent types of transporters for B uptake have beenidentified: NIP5;1 and BOR1 in Arabidopsis (Arabi-dopsis thaliana; Takano et al., 2008; Reid 2014). NIP5;1 isa member of the major intrinsic protein family, be-longing to subgroup II of nodulin 26-like intrinsicprotein (NIP; Takano et al., 2006; Mitani et al., 2008). Itfunctions as a boric acid channel (Takano et al., 2006).On the other hand, BOR1 functions as a boric acid/borate exporter (Takano et al., 2002), which is similar tobicarbonate transporters in animal cells. In Arabi-dopsis, NIP5;1 and BOR1 are polarly localized at thedistal and proximal side of the plasmamembrane of theroot cells, respectively (Takano et al., 2010), and theircooperative transport is required for efficient and di-rectional uptake of B in the roots especially under aB-limited condition (Takano et al., 2008; Reid 2014).Homologs of Arabidopsis NIP5;1 and BOR1 have beenfunctionally characterized in other plant species such asrice (Oryza sativa), barley (Hordeum vulgare), rapeseed(Brassica napus), and maize (Zea mays; Nakagawa et al.,2007; Sutton et al., 2007; Chatterjee et al., 2014; Durbaket al., 2014; Hanaoka et al., 2014; Leonard et al., 2014;Zhang et al., 2017). Basically, they have a similar role inB uptake, although the expression patterns differ

1 This work was supported by Grant-in-Aid for Specially Pro-moted Research (JSPS KAKENHI grant no. 16H06296 to J.F.M. and15H04469 to N.Y.).

2 Address correspondence to maj@rib.okayama-u.ac.jp.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Jian Feng Ma (maj@rib.okayama-u.ac.jp).

J.F.S., N.Y., R.F.S., and J.F.M. conceived and designed the experi-ments; J.F.S., X.W.L., and J.F.M. performed most of the experiments;N.Y. performed the immunostaining experiment; K.Y. performedwestern blot; J.F.S., N.Y., and J.F.M. analyzed data and wrote thearticle.

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somewhat within plant species. Recently, two maizemutants (tls1 and rte) defective in vegetative and in-florescence development were also found to be causedby loss of function of genes homologous to NIP5;1 andBOR1, respectively (Chatterjee et al., 2014; Durbaket al., 2014), highlighting the importance of B in plantgrowth and development. In addition, some PIPs(plasma membrane intrinsic proteins) such as OsPIP2;4and OsPIP2;7 in rice (Kumar et al., 2014), ZmPIP1 inmaize (Dordas and Brown, 2001), and HvPIP1;3 andHvPIP1;4 in barley (Fitzpatrick and Reid, 2009), havebeen implicated in B uptake. However, their exact roleand contribution to B-uptake are still unclear.

On the other hand, differential expression level ofBOR1 homolog (Bot1/BOR2) was involved in toleranceto B toxicity in barley (Reid 2007; Sutton et al., 2007).B-toxicity tolerant cultivar shows higher expression ofBot1/BOR2 than B-toxicity sensitive cultivar of barleydue to increased gene copy number. Different fromBOR1, Bot1/BOR2 in barley plays an important role inexcluding B from the cytosol to the rhizospheresprobably due to different cellular localization (Takanoet al., 2002; Sutton et al., 2007). This is similar toAtBOR4in Arabidopsis, which overexpression resulted in in-creased tolerance to B toxicity (Miwa et al., 2007). Inaddition to NIP5;1, HvLsi1, a silicon transporter inbarley (Chiba et al., 2009), was found to be involvedin the B uptake and its differential expression is re-sponsible for genotypic difference in B-toxicity toler-ance (Schnurbusch et al., 2010).

After B uptake mediated by NIP5;1 and BOR1, it willbe loaded to the xylem, followed by translocation fromthe roots to the shoots by transpirational mass flow(Marschner 2012). For a long time, it has been believedthat the distribution of B in the shoots is mainly deter-mined by relative transpiration rather than demand(Shelp et al., 1995). However, on the other hand, it wasobserved that B was preferentially delivered to devel-oping tissues inmany plant species such as Arabidopsis(Takano et al., 2001), sunflower (Helianthus; Matoh andOchiai, 2005), wheat (Triticum aestivum; Huang et al.,2001), and broccoli (Brassica oleracea, var. italica;Marentes et al., 1997), which have very low or notranspiration. Therefore, there must be a system forsuch preferential distribution of B especially underB-limited condition, but for a long time it has been amystery on howB taken up by the roots is preferentiallydistributed to the developing tissues. Recently, nodes ingramineous plants were found to play an importantrole in distribution of mineral elements (Yamaji andMa, 2014; 2017). Several transporters involved in theintervascular transfer from enlarged vascular bundle todiffuse vascular bundle for preferential distributionhave been identified, but most transporters localized inthe nodes have not been functionally characterized.Our genomewide transcriptional analysis with ricenode found that a member of nodulin 26-like intrinsicprotein (NIP), OsNIP3;1, was highly expressed in thenode (Yamaji et al., 2013). OsNIP3;1 is the closest ho-molog of AtNIP5;1 (Mitani et al., 2008). It is localized to

the plasma membrane and also permeable to boric acidin yeast (Saccharomyces cerevisiae; Hanaoka et al., 2014)as well as arsenite when expressed in Xenopus oocytes(Ma et al., 2008). Knockout of this gene resulted in re-tarded growth (Liu et al., 2015). It has been speculatedthat OsNIP3;1 is involved in B uptake, mobility, anddistribution among shoot tissues (Hanaoka et al., 2014;Liu et al., 2015), but the exact role of this gene is un-known. By detailed functional analysis, we found thatOsNIP3;1 was highly expressed in the rice nodes anddid not contribute to B uptake and translocation. Itsmain role is to preferentially deliver B to the developingtissues. Furthermore, we found that B distribution isregulated at both the transcriptional and protein levels.

RESULTS

OsNIP3;1 Showed Higher Expression in the Nodes

Although the expression pattern of OsNIP3;1 wasinvestigated in previous studies (Hanaoka et al., 2014;Liu et al., 2015), they only focused on the roots andshoots. However, we found that the expression washigher in the nodes than in the roots and shoots at boththe vegetative and reproductive stage (Fig. 1, A and B).Especially at reproductive stage, the expression in theuppermost node I of rice grown in rice field was muchhigher than that in other organs (Fig. 1B). At the vege-tative growth stage, the expression in the roots andshoot basal regions including basal nodes wereup-regulated by B deficiency, but that in the leaves wasunaffected (Fig. 1A). The expression of OsNIP3;1 in theroots and shoot basal regions was down-regulated by44% and 47%, respectively, at high external B concen-trations compared with -B, but the expression level wassimilar at 3 mM and 18 mM B (Fig. 1C). A time-courseexperiment showed that the expression of OsNIP3;1 inthe roots and shoot basal regions responded quickly toexternal B; at 30 min after exposure to 3 mM B, the ex-pression level of OsNIP3;1 in the shoot basal region wasreduced to one-half (Fig. 1D). The expression level was notfurther decreased after exposure to B for 2 h and longer.

OsNIP3;1 Protein was Mainly Localized at the XylemRegion of the Node

To investigate tissue and cell specificity localizationof OsNIP3;1 protein, we performed immunostainingusing an antibody against OsNIP3;1. At the vegetativestage, the signal was detected in all cells of the root tips(2 mm from root tip) and root mature region (20 mmfrom tip). Furthermore, it showed polar localization atthe distal side (Fig. 2, A and B). A stronger signal wasobserved in the basal node and leaf sheath (Fig. 2, C toE). At the basal node, OsNIP3;1 was localized at thexylem parenchyma cells including transfer cells of en-larged vascular bundle (Fig. 2C). In the leaf sheath,OsNIP3;1 was localized at the xylem region (Fig. 2, Dand E), but signal was very weak in the leaf blade

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(Fig. 2F). Furthermore, OsNIP3;1 also showed polarlocalization at the side facing toward the vessels in allxylem cells (Fig. 2, C to E).At the reproductive growth stage, a strong signal was

observed in the xylem parenchyma cells of enlargedvascular bundles of node I (Fig. 2, G and H). A weaksignal was also observed in the phloem region of diffusevascular bundle (Fig. 2I). Signal was also observed inthe xylem region of the peduncle (Fig. 2J). In these tis-sues, OsNIP3;1 also showed polar localization at thesides facing toward the xylem vessels. No signals weredetected at the nodes of two independent knockoutlines (Fig. 2, K and L), indicating the specificity of thisantibody against OsNIP3;1.

Rapid Degradation of OsNIP3;1 Protein in Response toHigh External B

To investigate the response of OsNIP3;1 protein toexternal B, we traced the protein change after theB-deficient plants were transferred to B solution using

the same timescale for the transcript experiment (Fig. 3).At the basal node and leaf sheath of young seedlings,OsNIP3;1 signal became weak after exposure to B so-lution for 2 h (Fig. 3, C, H, and M). At 12 h afterthe exposure, the signal completely disappeared. Thisresult is consistent with western blot and coimmunos-taining results (Supplemental Figs. S1 and S2).Western-blot analysis detected two bands at approximately50 kD and 75 kD, which were supposed to be a dimerand trimer of OsNIP3;1 (Supplemental Fig. S1). Thesignal of OsNIP3;1 protein disappeared after exposureto B solution for 12 h (Supplemental Fig. S1). Coim-munostaining showed that the signal of Lsi6, a Sitransporter (Yamaji et al., 2008), did not respond to highB (Supplemental Fig. S2), but that of OsNIP3;1 wasdegraded upon B exposure (Supplemental Fig. S2).

Conversely, when B-sufficient plants were exposedto a solution free of B, OsNIP3;1 was detected in thebasal node and leaf sheath at d 1 (Fig. 4). The signalbecame stronger at d 2 and thereafter. These resultsindicate that OsNIP3;1 protein was degraded or

Figure 1. Expression pattern of OsNIP3;1 in rice. A, Expression of OsNIP3;1 in different organs of plants with or without B atvegetative growth stage. Seedlings (22-d-old, cv Hwayoung) were cultured in a solution containing 0 mM (2B) or 3 mMB (+B) for7 d. Different organswere sampled for expression analysis. B, Expression ofOsNIP3;1 in different organs of rice grown in a field atdifferent growth stage. Different organs of rice (cv Nipponbare) grown in a paddy field were sampled at different growth stageshown. C, Effect of different B concentration on OsNIP3;1 expression. Seedlings (32-d-old, cv Hwayoung) precultured in B-freesolution for 3 d, were exposed to a solution containing 0mM, 3mM, or 18mMof B for 24 h. Different organs including roots, shootbasal region, and shoots were sampled. D, Time-dependent expression ofOsNIP3;1 in response to external B. Seedlings (16-d-old, cvHwayoung) precultured inB-free solution for 7 d,were exposed to 3mMB for different times indicated, and the roots, shoot basal region,and shoots were sampled. The expression level was determined by real time RT-PCR. HistoneH3 and Ubiquitin were used as internalstandards. Expression relative to the roots is shown. Data are means 6 SD (n = 3). Asterisk shows significant difference at P , 0.05between +B and 2B. Different small letter indicates significant difference at P , 0.05 by Duncan’s test.

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synthesized in response to external B concentration.OsNIP3;1 in the roots and node I was also degraded inresponse to high B (Supplemental Figs. S3 and S4).

Phenotypic Analysis of OsNIP3;1 Knockout Lines

To examine the role ofOsNIP3;1 in B-transport in rice,we obtained two independent knockout lines withT-DNA insertion (Supplemental Fig. S5, A and B).When grown at 0 mMand 0.3 mMB for 30 d, the growthof two mutants were severely inhibited compared withtheir wild types (Supplemental Fig. S5C). However, at3 mM B, the growth became similar between mutantsand wild types. This phenotype is similar to previousstudies using RNAi lines and a different mutant, dte1(Hanaoka et al., 2014; Liu et al., 2015). We further in-vestigated the effect of OsNIP3;1 knockout on the de-velopment of individual leaf. For this purpose, plants

precultured with 3 mM B were transferred to a solutioncontaining 0 mM or 3 mM B and individual leaves werecompared between wild types and mutants after 11 d.At 0 mM B, the young leaves (leaf 6 and leaf 7) thatappeared after transfer to –B solution were muchsmaller in the mutant than the wild type (Fig. 5, A andB; Supplemental Fig. S6A), showing chlorosis and ne-crosis at the tips, which are typical B-deficiency symp-toms. However, at 3mMB, the growth of fully expandedyoung leaf (leaf 6) did not differ between wild type andmutant (Fig. 5, C and D; Supplemental Fig. S6D).

We also compared B concentration and accumulationin different leaves. At 0 mM B, the concentration of B ineach leaf did not differ betweenwild types andmutants(Supplemental Figure S6B), but the B-accumulation inleaf 6 and leaf 7 was significantly reduced in the mu-tants compared with the wild types (Supplemental Fig.S6C). As a result, the distribution ratio of B in youngleaves (leaf 6 and leaf 7) was decreased in the mutants,

Figure 2. Tissue and cell specificity of localization of OsNIP3;1. Immunostaining using an antibody against OsNIP3;1 wasperformed in root tips (A), root mature region (B), basal node (C), leaf sheath (D and E), and leaf blade (F) of young seedlings (wildtype), and node I (G to I) and peduncle (J) of plants (wild type) and node I (K and L) of two knockout lines at heading stage. Redcolor shows signal fromOsNIP3;1 and blue/cyan color from cell wall autofluorescence. Insets of (A) and (B), (E), (H), and (I) are amagnified image of the yellow box area in (A), (B), (D), and (G), respectively. The xylem and phloem region of the enlargedvascular bundle and the diffuse vascular bundle (EVx, EVp, DVx, and DVp) are indicated. Scale bars = 100 mm. DVB, diffusevascular bundle; EVB, enlarged vascular bundle; RV, regular vascular bundle in basal node. co, cortex cell; en, endodermis; ep,epidermis; ex, exodermis; p, phloem region; sc, sclerenchyma; v, xylem vessel.

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but that in the old leaf (leaf 4) was increased (Fig. 5E). At3 mM B, the difference in both B concentration and ac-cumulation in each leaf was not evident between WTsand mutants (Supplemental Fig. S6, E and F). There wasalso no difference in the distribution, uptake, and root-to-shoot translocation of B between wild types and mutants(Fig. 5F; Supplemental Fig. S7). These results indicate thatOsNIP3;1 is involved in the distribution of B to the de-veloping tissues under B-limited conditions, rather thanin B uptake by the roots and root-to-shoot translocation.

Stable Isotope-Labeling Experiment

To further support the above conclusion, we per-formed a short-term labeling experiment by using stableisotopes of B, 10B, and 11B. Plants precultured with 11Bwere exposed to a solution containing 0 mM or 3 mM 11Bfor 3 d and then subjected to a solution containing 0.3mM 10B together with rubidium (Rb) and strontium (Sr)for 10 h. Rb and Sr are known as markers of phloem andxylem transport (Kuppelwieser and Feller, 1991), re-spectively. The uptake and the root-to-shoot transloca-tion of 10B during 10 h was similar between WTs andmutants (Supplemental Fig. S8, A and B). There wasalso no difference in B uptake between B-sufficient and-deficient plants of each line. However, there was a clear

difference in the concentration and distribution of 10B insome leaves of plants pretreated with 2B between WTsand mutants. Knockout of OsNIP3;1 resulted in a lower10B-concentration in the shoot basal regions and theyoungest leaf (leaf 6; Fig. 6A). The distribution of 10B tothe older leaves (leaf 2 to leaf 4) increased, but that to theyoung leaf significantly decreased in the mutants (Fig.6B). However, neither the concentration nor the distri-bution of either Rb or Sr was altered in the mutants(Supplemental Fig. S9). In plants pretreated with suffi-cient B, decreased 10B concentration in the shoot basalregion was also found in the mutants, although the dif-ference was not as large as in plants pretreated with –B(Fig. 6C). No difference was found in the distribution of10B to different leaves between WTs and mutants pre-treated with B (Fig. 6D). There was also no difference inthe concentration and distribution of Rb and Sr betweenWTs and mutants pretreated with B (Supplemental Fig.S9, C, D, G, and H). These results further indicate thatOsNIP3;1 is required for preferential distribution of B tothe developing tissues under the B-limited condition.

Silicon Transporter Lsi1 Largely Contributes to B Uptake

OsNIP3;1was previously proposed to be involved inthe B uptake in rice (Hanaoka et al., 2014). However,

Figure 3. Response of OsNIP3;1 protein to B supply. Seedlings (16-d-old, cv Hwayoung) precultured in B-free solution for 7 dwere exposed to a solution containing 3 mMB. At different times indicated, both basal nodes (A to J) and leaf sheath (K to O) weresampled for immunostaining of OsNIP3;1. F to J, Magnified image of xylem area of EVB from above panel (A to E). Red colorshows signal from OsNIP3;1 and blue/cyan color from cell wall autofluorescence. Scale bars = 100 mm.

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our results showed that the contribution ofOsNIP3;1 tototal B uptake was negligible (Supplemental Figs. S7Aand S8A). This raises a question: which transporter is

responsible for B uptake in rice? Previous studiesshowed that Si transporter Lsi1 is also permeable toboric acid in Xenopus oocyte (Mitani et al., 2008) and

Figure 4. Response of OsNIP3;1 proteinlocalized in the basal node and leafsheath to B deficiency. Seedlings (16-d-old, cv Hwayoung) precultured in 3 mMB solution were exposed to B-free solu-tion. At d0, d1, d2, and d5, the basalnode (A to H) and leaf sheath (I to L)were sampled for immunostaining ofOsNIP3;1. Red color shows signal fromOsNIP3;1 and blue/cyan color showscell wall autofluorescence. E to H,Magnified image of xylem area of EVB inbasal nodes shown above panel. Scalebars = 100 mm.

Figure 5. Phenotypic analysis ofOsNIP3;1 knockout lines. A and D, Phenotype ofOsNIP3;1mutant at different B. Seedlings (17-d-old) of wild type (A and C) andmutant (B andD) preculturedwith 3mMBwere exposed to a solution containing 0mM (A and B)or 3 mM B (C and D). After 11 d, shoot basal region and individual leaf (leaf 2 to leaf 7, from old to young) were separated andphotographed. E and F, Distribution of B in different organs of two mutants (nip3;1-1 and nip3;1-2) and their corresponding wildtypes (WT1 andWT2) at 0 (E) and 3 mM B (F). Distribution was calculated based on B accumulation in different organs. Data aremeans 6 SD (n = 3). Asterisk shows significant difference at P , 0.05 between wild type and mutant. Scale bars = 10 cm.

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yeast (Schnurbusch et al., 2010). By using our lsi1 mu-tant, it was also reported that the mutant contained lessB compared with its wild type (Bienert et al., 2008;Schnurbusch et al., 2010). To further investigate theinvolvement of Lsi1 in B uptake, we grew both lsi1mutant and its wild type at different B concentrations.At 0 mM B, the youngest leaf of the mutant showed atypical B-deficiency symptom, which is characterized bythe retarded growth of the leaf meristem (SupplementalFig. S10). However, such a symptomwas not observed inthe wild type. At 0.3 mMand 3 mMB, the growth becamesimilar between the wild type and the lsi1 mutant(Supplemental Fig. S10). The B concentration in the shootwas significantly lower in the mutant than in the wildtype at 0.3 mMand 3 mMB (Fig. 7A), but the difference inthe root B concentrationwasnot large (Fig. 7B). The total Buptake of lsi1 was only 49% and 44% of wild type, re-spectively, at 0.3 mM B and 3 mM B (Fig. 7C). More than94% of total B was translocated to the shoots, but therewas no difference in the root-to-shoot translocation of Bbetween wild type and lsi1 (Fig. 7D).Because the expression level of Lsi1was much higher

than that ofOsNIP3;1 in the roots (Ma et al., 2008), thereis a possibility that the contribution of OsNIP3;1 to

B uptake may be overlapped by Lsi1. Because Lsi1is mainly expressed in mature root but not in root tip(Yamaji andMa, 2007), we therefore performed a short-term (6 h) uptake experiment using 10B and comparedthe 10B concentration of the root tips (0 to 0.5 cm)between osnip3;1 mutants and their wild types.The 10 B concentration in the root tips was signifi-cantly lower in the mutants than in the wild types(Supplemental Fig. S11). This result indicates thatOsNIP3;1locally contributes to B uptake in the root tips, althoughits contribution to total B uptake is small.

We also investigated the response of Lsi1 expressionto different B concentrations. Unlike OsNIP3;1, the ex-pression of Lsi1 was constant and not affected by theexternal B concentrations (Supplemental Fig. S12).

DISCUSSION

OsNIP3;1 Is Responsible for Preferential Distribution of Bto Developing Tissues

Previous physiological studies have shown that Btaken up by the roots is preferentially delivered to thedeveloping tissues such as new leaves and reproductive

Figure 6. Concentration and distribution of 10B in different organs. A and C, 10B concentration in different organs of OsNIP3;1knockout lines (osnip3;1-1 and osnip3;1-2) and their corresponding wild types (WT1 and WT2). B and D, Distribution of 10B indifferent organs. Seedlings (22-d-old) preculturedwith 3 mM 11Bwere exposed to a solution containing 0mM (A and B) or 3mM 11

B (C and D) for 3 d. These seedlings were then exposed to a solution containing 0.3 mM 10B. After 10 h, each organwas separatelyharvested and subjected to 11B and 10B determination by ICP-MS. Data are means 6 SD (n = 3). Asterisk shows significant dif-ference at P , 0.05 between wild type and mutant.

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organs for their active growth (Marentes et al., 1997;Takano et al., 2001; Huang et al., 2001; Matoh and Ochiai,2005), but the molecular mechanism underlying thispreferential distribution is poorly understood. In thisstudy, we found that OsNIP3;1 localized in the nodes isrequired for this preferential distribution of B in rice.Nodes are the hub of distribution of mineral elements(Yamaji andMa, 2014), which have a complexed but wellorganized vascular system. We found that OsNIP3;1 ishighly expressed in the nodes at both the vegetative andreproductive growth stage (Fig. 1, A and B). In the nodes,OsNIP3;1 is mainly localized in the xylem parenchymacells (Fig. 2, C and G). Furthermore, it is polarly localizedat the side, facing toward the xylem vessel (Fig. 2, C, G,and H). Knockout of OsNIP3;1 resulted in decreaseddistribution of B to the developing tissues under B-limitedcondition (Fig. 6B). These results together indicate thatOsNIP3;1 is involved in unloading B in the xylem of en-larged vascular bundle (Fig. 8B), which is the first step forthe intervascular transfer. In addition, OsNIP3;1 is alsolocalized in the phloem region of diffuse vascular bundlesof node I (Fig. 2I). This also facilitates preferential distri-bution of B to the developing tissues by loading B into thephloem companion cells (Fig. 8B). However, becauseOsNIP3;1 is a passive channel for boric acid, a cooperativeefflux transporter for the intervascular transfer of B is re-quired as a driving force (Yamaji and Ma, 2014; Yamajiet al., 2015; Yamaji and Ma, 2017), which remains to beidentified. Localization of OsNIP3;1 is also observed inthe xylem region of leaf sheath and peduncle (Fig. 2, Dand J), indicating its similar role in unloading B from thexylem in these organs. A higher B ratio of leaf blade/sheath in RNAi lines observed previously (Hanaoka et al.,2014) is probably the result of decreased unloading of Bwithin the leaf sheath.

In Arabidopsis, AtNIP6;1 was implicated in thepreferential distribution of B to growing shoot tissues

(Tanaka et al., 2008).AtNIP6;1 is predominantly expressedin nodal regions of shoots, especially in the phloemregion of vascular tissues. Knockout of AtNIP6;1resulted in lower B concentration in the young rosetteleaves (Tanaka et al., 2008). Based on this evidence, itwas suggested that AtNIP6;1 is involved in xylem-phloem transfer of B at the nodal regions. These find-ings indicate that Arabidopsis has a different system forpreferential distribution of B from rice. In fact, the nodestructure is totally different between Arabidopsis andrice (Yamaji and Ma, 2017). Unlike rice, Arabidopsisnode does not have developed vascular system.

OsNIP3;1 Is Regulated at Both Transcriptional andProtein Level

The expression of OsNIP3;1 was down-regulated byB supply in the roots (Hanaoka et al., 2014; Liu et al.,2015). In this study, we found that expression ofOsNIP3;1 in the nodeswas also rapidly down-regulatedin response to B supply (Fig. 1C). A time-course ex-periment showed that the expression level in the nodesdecreased half at 30 min after exposure to B (Fig. 1D)andmaintained at a constant level after 2 h.AtNIP5;1 inArabidopsis was also down-regulated by B (Takanoet al., 2006). The 59 UTR was involved in the regulationof AtNIP5;1 expression (Tanaka et al., 2011). It seemsthat high B triggers AtNIP5;1 mRNA degradation.Furthermore, it was recently found that the minimumopen reading frame, AUG-stop induced B-dependentribosome stalling and mRNA degradation of AtNIP5;1(Tanaka et al., 2016). The 59 UTR is highly conservedbetween AtNIP5;1 and OsNIP3;1 (Tanaka et al., 2011).This suggests that similar mechanism is involved in theregulation of OsNIP3;1 in rice although further confir-mation is required in future.

Figure 7. Concentration, uptake, androot-to-shoot translocation of B in thelsi1 knockout line and its wild type. Aand B, B concentration in the shoots(A) and roots (B). C and D, Uptake (C)and root-to-shoot translocation (D).Seedlings (6-d-old) of the lsi1mutant andits wild type (cv Oochikara) were grownin a nutrient solution containing 0 mM,0.3 mM, or 3 mMB. After 14 d, the plantswere harvested and subjected to Bdetermination by ICP-MS. Data aremeans 6 SD (n = 3). Asterisk shows sig-nificant difference at P , 0.05 betweenwild type and mutant.

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The OsNIP3;1 protein localized in the nodes alsoshowed rapid response to external B change; the pro-tein was rapidly degraded in response to high B (Fig. 3;Supplemental Figs. S1 and S2), whereas it was gener-ated in response to B deficiency (Fig. 4). At 12 h afterexposure to high B, OsNIP3;1was completely degradedalthough its transcript was still detected (Figs. 1D andFig. 3; Supplemental Figs. S1 and S2). This means thatabsence of OsNIP3;1 protein at high B is not solely dueto its decreased transcript accumulation, but to anotherunknown mechanism for the degradation. BOR1 inArabidopsis also undergoes degradation in response tohigh B (Takano et al., 2010). BOR1 is transferred viaendosomes to the vacuole for degradation upon high Bsupply (Takano et al., 2005) and Tyr residues in thelarge loop region of BOR1 are required for the vacuolarpathway (Takano et al., 2010). Unlike BOR1, we did notobserve dotlike structures of OsNIP3;1 in the cyto-plasm during degradation process in the time-courseexperiment (Fig. 3), suggesting that a different mecha-nism for degradation of OsNIP3;1 is involved.

Different Regulation of B Accumulation in Riceand Arabidopsis

In natural systems, B concentrations vary from van-ishingly small to very high (Tanaka et al., 2011; Reid2014), but the range between deficiency and toxicity of

B is very narrow. Therefore, plants must have a systemto maintain B homeostasis by regulating the uptake,mobilization, distribution, and sequestration. In Ara-bidopsis, B homeostasis seems to be maintainedthrough regulating B uptake mediated by NIP5;1 andBOR1. NIP5;1 undergoes transcriptional regulation(Takano et al., 2010; Tanaka et al., 2011), whereas BOR1is regulated at posttranscriptional regulation in re-sponse to external B change (Takano et al., 2005). Riceseems to have a different mechanism for B homeostasis.OsNIP3;1, the closest homolog of AtNIP5;1, did notcontribute to total B uptake (Supplemental Figs. S7Aand S8A), but a Si transporter Lsi1 is largely involved inB uptake in rice roots (Fig. 7C). Lsi1 is localized at thedistal side of exodermal and endodermal cells of themature root region (Ma et al., 2006), which is respon-sible for transport of B from external solution to the rootcells (Fig. 8A). However, unlike AtNIP5;1, the expres-sion of Lsi1 was not affected by external B level(Supplemental Fig. S12). A homolog of BOR1 in rice,OsBOR1, was reported as an efflux B transporter(Nakagawa et al., 2007), although it is not clear whetherOsBOR1 is localized at the proximal side of exodermaland endodermal cells for cooperative transport withLsi1. The accumulation of OsBOR1 transcripts was alsonot affected by B deprivation, but the protein seems tobe enhanced (Nakagawa et al., 2007). Therefore, there isa possibility that the B uptake is regulated by OsBOR1at posttranscriptional level. However, we found that

Figure 8. Schematic presentation of role of OsNIP3;1 in uptake and distribution of B in rice. A, Role of OsNIP3;1 in B uptake.OsNIP3;1 polarly localized at the distal side of cells in the root tips locally contributes to B influx into the cells. In the root matureregion, Lsi1 polarly localized at the distal side of exodermis and endodermis mediates B influx to the root cells. OsBOR1 isproposed to be involved in B efflux from the root cells. B, Role of OsNIP3;1 in preferential distribution of B to developing tissues.OsNIP3;1 polarly localized at the xylem transfer cells of enlarged vascular bundle mediates unloading of B from the xylem forintervascular transfer. Efflux B transporter for the intervascular transfer is unidentified. OsNIP3;1 located at the phloem cell ofdiffuse vascular bundle also facilitates B influx to the phloem for preferential distribution. OsNIP3;1 is regulated at both tran-scriptional and protein level in response to environmental B change.

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there was no large difference in B uptake betweenB-sufficient and -deficient rice plants (SupplementalFig. S8A), suggesting that unlike Arabidopsis, B uptakeis not regulated by external B supply. By contrast, it islikely that B homeostasis in rice is regulated at thedistribution step mediated by OsNIP3;1 in the nodes.Under the B-limited condition, OsNIP3;1 mediatespreferential distribution to developing tissues to meettheir requirement (Figs. 6B and 8B). However, underthe high B condition, OsNIP3;1 is rapidly degraded toavoid excess B to be delivered to the developing tissues.One possible explanation for such different regulationbetween rice and Arabidopsis is that Lsi1 responsiblefor Si and B uptake shows relatively wide transportsubstrates and rice accumulates much more Si than B(Ma and Yamaji, 2015), therefore it is difficult to controlB influx at the uptake step. Moreover, Arabidopsis doesnot have Si channel homologous to rice Lsi1 in the ge-nome (Ma and Yamaji, 2015). This regulation mecha-nism is similar to that for Mn in rice (Yamaji et al., 2013;Shao et al., 2017). OsNramp3 in the node required forMn distribution, rather than OsNramp5 and OsMTP9in the roots for Mn uptake, is regulated at the proteinlevel in response to environmental Mn change (Shaoet al., 2017).

In conclusion, OsNIP3;1 mainly localized in thenodes mediates intervascular transfer of B by unload-ing B from xylem of enlarged vascular bundles and isrequired for preferential distribution of B to the devel-oping tissues. OsNIP3;1 is regulated at both transcrip-tional and protein level in response to externalB-concentration change.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Two T-DNA insertion mutant lines of OsNIP3;1 (4A-01956/nip3;1-1 and2A-00764/nip3;1-2) from RISD (http://cbi.khu.ac.kr/RISD_DB.html) andtheir wild-type rice (Oryza sativa; WT1; cv Dongjin, WT2; cv Hwayoung);lsi1mutant and its wild type (cv Oochikara), were used in this study. Seedswere soaked in water for 2 d at 30°C in the dark and then the geminatedseeds were transferred to nylon nets floating on a solution containing0.5 mM CaCl2 in a 1.2-L pot and grown for 5 d. The seedlings were trans-ferred to a 1/2 Kimura B-nutrient solution (pH 5.6) containing the fol-lowing macronutrients: MgSO4 (0.28 mM), (NH4)2SO4 (0.18 mM), Ca(NO3)2(0.18 mM), KNO3 (0.09 mM), and KH2PO4 (0.09 mM); and micronutrients:Fe-EDTA (20 mM), H3BO3 (3 mM), MnCl2 (0.5 mM), CuSO4 (0.2 mM), ZnSO4(0.4 mM), and (NH4)6Mo7O24 (1 mM). The solution was renewed every 2 d.All experiments were conducted in a greenhouse at 25°C to 30°C undernatural sunlight with three replicates.

Gene Expression Pattern of OsNIP3;1 and OsLsi1

To investigate the expression of OsNIP3;1 in different organs at vegetativegrowth stage, seedlings (22-d-old, cv Hwayoung) were cultured in a solutioncontaining 0 (2B) or 3 mMB (+B) for 7 d. Different organs including roots, shootbasal region, and different leaves were sampled and immediately frozen inliquid nitrogen until extraction of RNA. The effect of different B concentrationson OsNIP3;1 expression was investigated by exposing seedlings (32-d-old, cvHwayoung) precultured in B free solution for 3 d, to a solution containing 0mM,3 mM, and 18 mM B. After 24 h, roots, shoot basal region, and shoots weresampled. Furthermore, a time-course experiment of OsNIP3;1 expression wasperformed by exposing seedlings (16-d-old, cv Hwayoung) precultured in

B-free solution for 7 d, to a solution containing 3 mM B. At times indicated, theroots, shoot basal region, and shoots were sampled. The expression ofOsNIP3;1was also investigated in different organs at different growth stages in ricegrown in a paddy field using the same samples described previously (Yamajiet al., 2013).

Effect of different B concentrations on Lsi1 expression was investigated inplants (27-d-old, cv Hwayoung) exposed to a solution containing 0 mM, 3 mM,and 18 mM B. After exposure for 24 h, the roots were sampled for RNAextraction.

Total RNA of samples harvested was extracted by using an RNeasy PlantMini Kit (Qiagen), followed by DNase I treatment. Conversion to cDNA wasperformed using the protocol supplied by the manufacturer of SuperScript II(Invitrogen). Specific cDNAs were amplified by Sso Fast EvaGreen Supermix(Bio-Rad). The expression was determined by quantitative RT-PCR usingprimers (F1) 59-AATCAGTCGTGTCATCCTTGGG-39 (forward) and (R1)59-CCCATCTACGTGAATGGACCG-39 (reverse) for OsNIP3;1, 59-GCCAGCAA-CAACTCGAGAACAA-39 (forward) and 59-CATGGTAGGCATGGTGCCGT-39(reverse) for Lsi1 on CFX384 (Bio-Rad). HistoneH3 and Ubiquitin were used as in-ternal standards with primers 59-AGTTTGGTCGCTCTCGATTTCG-39 (forward)and 59-TCAACAAGTTGACCACGTCACG-39 (reverse) for HistoneH3,59-GCTCCGTGGCGGTATCAT-39 (forward) and 59-CGGCAGTTGA-CAGCCCTAG-39 (reverse) for Ubiquitin. The relative expression wasnormalized based on these two genes by ΔΔCt method using the CFXManager software (Bio-Rad).

Immunohistological Staining of OsNIP3;1

The synthetic peptide C-LRDENGETPRPQRSFRR (positions 293 to 309 ofOsNIP3;1)was used to immunize rabbits to obtain antibodies against OsNIP3;1.The obtained antiserum was purified through a peptide affinity column beforeuse. Different organs including roots, basal node, leaf sheath, blade, node I, andpeduncle of both wild types and mutants were used for immunostaining ofOsNIP3;1 protein as described previously (Yamaji and Ma, 2007; Yamaji et al.,2008).

To investigate the response of OsNIP3;1 protein to B supply, seedlings (16-d-old, cv Hwayoung) precultured in B-free solution for 7 d were exposed to asolution containing 3 mM B. At different times indicated, basal nodes and leafsheath were sampled for immunostaining of OsNIP3;1 as described above. Totrace the response of OsNIP3;1 protein to B deficiency, seedlings (16-d-old, cvHwayoung), precultured in 3 mM B-solution, were exposed to B-free solution.At d0, d1, d2, and d5, the basal nodes and leaf sheath were sampled forimmunostaining of OsNIP3;1. Response of OsNIP3;1 protein localized in theroots in response to high B was examined in seedlings (5-d-old) of both wildtype (cv Donging) and osnip3;1 mutant, which were exposed to a solutioncontaining 0 mM or 10 mM B. After 12 h, different root segments (2 mm and20 mm from the root tip) were sampled for immunostaining. To compareOsNIP3;1 in node I between B-sufficient and -deficient plants, plants werehydroponically cultivated until the heading stage. Before sampling node I forimmunostaining, the plants were subjected to 0 mM or 3 mM B for 3 d. Obser-vation was made with a confocal laser scanning microscope (TCS SP8x; LeicaMicrosystems).

For coimmunostaining of OsNIP3;1 and Lsi6 as a positive control, weused a Zenon Rabbit IgG Labeling Kit (Molecular Probes) in accordancewith the manufacturer’s instructions. Seedlings (15-d-old) of both the WT2(cv Hwayoung) and the mutant (osnip3;1-2) were precultured in B-freesolution for 7 d, followed by exposing to a solution containing 3 mM B.Basal nodes were sampled for coimmunostaining at 0 h, 2 h, 12 h, and 24 hafter exposure to B. Fluorescence signals were observed with a confocallaser-scanning microscopy (TCS SP8x; Leica Microsystems). Signal in-tensities of each channel were measured by LAS X Software (LeicaMicrosystems) and the ratio relative to control 2B condition (0 h) werecalculated in each xylem region of the enlarged vascular bundles. A totalof 12 to approximately 25 regions from two to approximately four sectionswere used for the calculation.

Western-blot Analysis

For western-blot analysis, seedlings (15-d-old) of both the WT2 (cvHwayoung) and themutant (osnip3;1-2) were precultured in a B-free solution for7 d, followed by exposure to a solution containing 3 mMB. At different times asdescribed above, shoot basal regions (1 cm from the root to shoot junction) weresampled and subjected to protein extraction as described previously (Mitani

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et al., 2009). The microsome fraction was obtained by ultracentrifugation at100,000g for 40 min. The same amounts (10 mg) of microsome protein weresubjected to SDS-PAGE using 5% to 20% gradient polyacrylamide gels(e-PAGEL, ATTO; http://www.atto.co,jp) and subsequently to immune-blotting. The membrane was treated with OsNIP3;1 antibody (1:500 dilu-tions). Anti-rabbit IgG (H+L) HRP Conjugate (1:10,000 dilution; Promega;http://www.promega.com) was used as a secondary antibody. Themembranewas finally subjected to Coomassie Brilliant Blue staining. The ECL PlusWestern Blotting Detection System (GE Healthcare; http://www.gehealthcare.com) was used for detection via chemiluminescence.

Phenotypic Analysis of OsNIP3;1 Knockout Lines andlsi1 Mutant

Forphenotypic analysis, seedlings (17-d-old) of both knockout lines and theirwild types were exposed to various B-concentrations (0 mM, 0.3mM, and 3mM).After exposure for 11 d, the roots were washed with 5 mM CaCl2 solution forthree times and separated from the shoots. In above-ground parts, shoot basalregion (0 cm to 0.5 cm from the root-shoot junction) and different leaves (leaf2 to leaf 7) of one plant each were separately harvested for B determination andphotographed. To observe the prolonged effect of B deficiency on plant growth,some seedlings were exposed to –B solution for 30 d and then photographed.

To compare growth and B uptake between the lsi1mutant and its wild type(cv Oochikara) under different B concentrations, the seedlings (6-d-old) wereexposed to a nutrient solution containing 0 mM, 0.3 mM, and 3 mM B for 14 d(one plant each for different B concentrations, three replicates). After the plantswere photographed, the roots were washed with 5 mM CaCl2 three times,separately from the shoots.

Labeling Experiment with the Stable Isotope 10B

To investigate the distribution pattern of B, seedlings (22-d-old) of two in-dependent mutants and their wild types precultivated in a nutrient solutioncontaining 1 mM Si and 3 mM 11B (99% enriched boric acid; Cambridge IsotopeLaboratories) were exposed to 0 mM (2B) or 3 mM 11B (+B) for 3 d. Theseseedlings were then exposed to 0.3mM 10B (99% enriched boric acid; CambridgeIsotope Laboratories) with 1 mMRb and Sr in a nutrient solution. After 10 h, theroots were washed with 5 mM CaCl2 solution three times. Roots, shoot basalregion (0 cm to 0.5 cm from the root-shoot junction), and different leaves (leaf2 to leaf 6) were separately sampled and subjected to determination of 10B asdescribed below. Leaf 1 was too small to be sampled.

Determination of 10B Concentration in Root Tips

To investigateBuptake in root tips, seedlings (24-d-old) of twoknockout linesand their wild types precultured in 1/2 Kimura B-solution containing 3 mM B,were exposed to B-free (2B) solution for 3 d. These seedlings were then exposedto 0.3 mM 10B. After 6 h, the roots were washed with 5 mM CaCl2 solution forthree times and the root tips (0 cm to 0.5 cm, 20 to 30 root tips for one replicate)were excised by a razor. Their fresh weight was immediately recorded. Afterdigestion, 10B concentration in the root tips was determined as described below.

Measurement of B Concentration

All samples harvested were dried at 70°C in an oven for 3 d. The sampleswere digested with HNO3 (60%) and H2O2 mixture (HNO3: H2O2=1:1) at atemperature up to 110°C in 15-mL plastic tubes. After dilution, the concentra-tion of B as well as Rb and Sr was determined by inductively coupled plasmamass spectrometry (ICP-MS, 7700X; Agilent Technologies). An isotope modewas used for quantification of 10B and 11B in the digest solution.

Statistical Analysis of Data

Data were analyzed using Student’s t test and Duncan’s test. Significancewas defined as P , 0.05.

Accession Number

Sequence data from this article can be found in the GenBank/EMBL data-bases under accession no. AB856420 for NIP3;1.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Western-blot analysis of OsNIP3;1 in nodes ofwild-type rice and mutant.

Supplemental Figure S2. Time-dependent degradation of OsNIP3;1 inresponse to high B.

Supplemental Figure S3. Response of OsNIP3;1 protein localized in theroots in response to high B.

Supplemental Figure S4. Response of OsNIP3;1 protein localized in node Iin response to high B.

Supplemental Figure S5. Position of T-DNA insertion (A) and isolation ofOsNIP3;1 mutants (B). Phenotype of osnip3;1 mutants at different B con-centrations (C).

Supplemental Figure S6. Dry weight, concentration, and content of B indifferent organs.

Supplemental Figure S7. Uptake and root-to-shoot translocation of B inosnip3;1 knockout lines and their wild types.

Supplemental Figure S8. Uptake and root-to-shoot translocation of 10B inosnip3;1 knockout lines and their wild types.

Supplemental Figure S9. Concentration and distribution of Rb and Sr indifferent organs of osnip3;1 knockout lines and their wild types.

Supplemental Figure S10. Phenotype of lsi1 knockout line and its wildtype at different B concentrations.

Supplemental Figure S11. 10B concentration in the root tips (0 cm to0.5 cm) of osnip3;1 mutants and their wild types.

Supplemental Figure S12. Response of Lsi1 expression to different Bconcentrations.

ACKNOWLEDGMENTS

We thank Junpei Takano for providing 11B and 10B compounds and SanaeRikiishi for ICP-MS determination.

Received November 13, 2017; accepted December 4, 2017; published December7, 2017.

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