Roles of BOR2, a Boron Exporter, in Cross Linking of … · BOR2 mutants were not signiﬁcantly different from those in wild-type plants, but the proportion of cross-linked RG-II

Roles of BOR2, a Boron Exporter, in Cross Linking ofRhamnogalacturonan II and Root Elongation under BoronLimitation in Arabidopsis1[W]

Kyoko Miwa*, Shinji Wakuta, Shigeki Takada, Koji Ide, Junpei Takano, Satoshi Naito, Hiroyuki Omori,Toshiro Matsunaga, and Toru Fujiwara

Creative Research Institution, Hokkaido University, Sapporo 001–0021, Japan (K.M.); Graduate School ofAgricultural and Life Sciences, The University of Tokyo, Tokyo 113–8657, Japan (K.M., H.O., T.F.); GraduateSchool of Agriculture, Hokkaido University, Sapporo 060–8589, Japan (S.W., S.T., K.I., J.T., S.N.); andAgricultural Research Center, National Agriculture and Food Research Organization, Tsukuba 305–8666,Japan (T.M.)

ORCID IDs: 0000-0001-7328-3628 (K.M.); 0000-0002-7474-3101 (J.T.); 0000-0001-7006-5609 (S.N.); 0000-0002-5363-6040 (T.F.).

Boron (B) is required for cross linking of the pectic polysaccharide rhamnogalacturonan II (RG-II) and is consequently essentialfor the maintenance of cell wall structure. Arabidopsis (Arabidopsis thaliana) BOR1 is an efflux B transporter for xylem loading ofB. Here, we describe the roles of BOR2, the most similar paralog of BOR1. BOR2 encodes an efflux B transporter localized inplasma membrane and is strongly expressed in lateral root caps and epidermis of elongation zones of roots. Transfer DNAinsertion of BOR2 reduced root elongation by 68%, whereas the mutation in BOR1 reduced it by 32% under low B availability(0.1 mM), but the reduction in shoot growth was not as obvious as that in the BOR1mutant. A double mutant of BOR1 and BOR2exhibited much more severe growth defects in both roots and shoots under B-limited conditions than the corresponding singlemutants. All single and double mutants grew normally under B-sufficient conditions. These results suggest that both BOR1 andBOR2 are required under B limitation and that their roles are, at least in part, different. The total B concentrations in roots ofBOR2 mutants were not significantly different from those in wild-type plants, but the proportion of cross-linked RG-II wasreduced under low B availability. Such a reduction in RG-II cross linking was not evident in roots of the BOR1mutant. Thus, wepropose that under B-limited conditions, transport of boric acid/borate by BOR2 from symplast to apoplast is required foreffective cross linking of RG-II in cell wall and root cell elongation.

Boron (B) is an essential trace element for plants(Warington, 1923). B deficiency and toxicity have neg-ative effects on plant growth and development. Insuf-ficient B availability affects the quality and quantity ofagricultural production (Shorrocks, 1997) through theinhibition of root elongation, leaf expansion, and fertility(for review, see Loomis and Durst, 1992; Marschner,1995; Dell and Huang, 1997; Miwa and Fujiwara,2010). A number of physiological studies suggest thatB deficiency affects cell elongation rather than cell divi-sion in the growing portions of plants (Dell and Huang,1997). There is also evidence that cross linking of the

pectic polysaccharide rhamnogalacturonan II (RG-II)in cell walls is the primary physiological role of B inplants (O’Neill et al., 2004).

When the B supply is limited, most B in plants islocalized in the cell wall. Matoh et al. (1993) isolated aB-polysaccharide complex from the cell walls of radish(Raphanus sativus) roots, which was later identified asan RG-II-B complex (Ishii andMatsunaga, 1996; Kobayashiet al., 1996; O’Neill et al., 1996). The importance of RG-IIin reproduction has been demonstrated through theanalysis of an Arabidopsis (Arabidopsis thaliana) mutantincapable of synthesizing CMP-3-deoxy-D-manno-2-octulosonic acid, an activated form of an RG-II-specificmonosaccharide (Kobayashi et al., 2011). Borate formsa cis-diol ester with apiosyl residues in one of the fourside chains of the two RG-II monomers to generate theRG-II-B dimer (Ishii et al., 1999). The Arabidopsis mutantmurus1 contains reduced levels of Fuc, a glycosyl residueof RG-II (O’ Neill et al., 2001), and has altered RG-IIstructure (Pabst et al., 2013). Compared with the wildtype, murus1 exhibits reduced cross linking of RG-II byborate and reduced leaf expansion under sufficientlevels of B (O’ Neill et al., 2001). The application of highconcentrations of B restores both the cross linking ofRG-II and leaf expansion in this mutant, suggestingthat the cross linking of RG-II by borate is essential fornormal leaf expansion (O’Neill et al., 2001).

1 This work was supported in part by a Grant-in-Aid for YoungScientists (A; grant no. 22688005 to K.M.), a Grant-in-Aid for ScientificResearch (S; grant no. 25221202 to T.F.), and a Grant-in-Aid for Sci-entific Research on Innovative Areas (grant nos. 25114502, 25114506,and 22119002 to K.M. and T.F.) from the Ministry of Education, Cul-ture, Sports, Science, and Technology of Japan and the NEXT pro-gram (grant no. GS001 to J.T.) from the Japanese Society for thePromotion of Science.

* Address correspondence to [email protected] 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:Kyoko Miwa ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.113.225995

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For B to be utilized by plants, it must be transportedfrom the soil and through the roots to the shoots. In ad-dition to passive diffusion across membranes (Marschner,1995; Dordas and Brown, 2000), B transport is facilitatedby boric acid channels, which are major intrinsic proteins.Takano et al. (2006) identified nodulin26-like intrinsicprotein5;1 (NIP5;1) in Arabidopsis as a boric acid channelrequired for efficient uptake into root cells underB-limited conditions. ArabidopsisNIP6;1, the gene mostsimilar to NIP5;1, contributes to the preferential distri-bution of B in young shoot tissues (Tanaka et al., 2008).

BOR1 was the first B transporter identified throughan analysis of the Arabidopsis bor1-1mutant that requireshigh levels of B, 30 and 100 mM B supply for normal leafexpansion and fertility, respectively (Noguchi et al., 1997;Takano et al., 2002). Shoot growth in bor1-1 plants isseverely inhibited under low-B conditions, but rootgrowth is not (Takano et al., 2001). Borate RG-II crosslinking in shoots is reduced in bor1-1 under the low-Bcondition, correlating with the reduced B concentrationsin total shoots and shoot cell wall fraction (Noguchiet al., 2003). The bor1-1mutant has no apparent defect inB uptake by roots but is impaired in efficient root-to-shoot translocation of the mineral under low-B condi-tions (Noguchi et al., 2000; Takano et al., 2002). BOR1encodes an efflux-type B transporter that is localized inthe plasma membrane and is expressed in various rootcells, including those in the endodermis (Takano et al.,2002, 2010). Thus, BOR1 is required for effective xylemloading under B-limited conditions, and BOR1 is alsolikely to be involved in the preferential distribution ofB to young leaves (Takano et al., 2001). Interestingly,BOR1 accumulation is regulated by B conditions at theposttranscriptional level, and the protein is recycled be-tween the plasmamembrane and endosomes under low-Bconditions. In addition, it is transported to the vacuole fordegradation when B levels are high (Takano et al., 2005,2010). Ubiquitination of the Lys-590 residue in BOR1 isrequired for this degradative process (Kasai et al., 2011).

There are six BOR1 paralogs in the Arabidopsis ge-nome. Overexpression of BOR4 confers high B tolerancein plants, suggesting that it is involved in B tolerancethrough the export of the mineral out of symplast(Miwa et al., 2007; Miwa and Fujiwara, 2011). BOR4 isstably accumulated in the plasma membrane at toxiclevels of B (Miwa et al., 2007).

Here, we report an investigation of the role of BOR2,the paralog most similar to BOR1, in B transport inArabidopsis. Based on analyses of the transport activity,expression, and the phenotypes of transfer DNA (T-DNA)insertion mutants, we propose a new function for an ef-flux B transporter in root cell elongation under B-limitedconditions.

RESULTS

B Transport Activity of BOR2 in Yeast

In the Arabidopsis genome, six genes are predictedto encode proteins with greater than 60% amino acid

sequence similarity to BOR1 (Arabidopsis GenomeInitiative, 2000; Nakagawa et al., 2007b). We obtainedfull-length complementary DNAs (cDNAs) for all sixparalogs. Based on the extent of amino acid sequencesimilarity to BOR1, we previously named the six paralogsAt3g62270, At3g06450, At1g15460, At1g74810, At5g25430,and At4g32510 as BOR2, BOR3, BOR4, BOR5, BOR6, andBOR7, respectively (Nakagawa et al., 2007b).

The paralog BOR2 is most similar to BOR1. The pre-dicted open reading frame (ORF) in the BOR2 cDNAconsists of 703 amino acids with 90% sequence identity toBOR1. BOR2 contains 10 to 11 putative transmembraneregions according to Aramemnon (http://aramemnon.botanik.uni-koeln.de/), as is the case for BOR1. BOR1and BOR2 reside on chromosomes 2 and 3, respectively,and correspond to regions of genome duplication (Arabi-dopsis Genome Initiative, 2000). Both BOR1 and BOR2contain 12 exons and 11 introns, and their exon-intronbreak points are identical.

BOR2 was expressed in a yeast (Saccharomyces cerevisiae)strain lacking the BOR1 homolog ScBOR1 (Takanoet al., 2007) under the control of the GAL1 promoter todetermine the B transport activity of BOR2. Cells atmidlog phase were harvested and incubated for 60 minin medium containing 0.1 or 0.5 mM boric acid. Fol-lowing incubation, the cells were washed twice in coldwater, boiled, and then centrifuged. The concentrationof B in the supernatant was determined to estimate theamount of soluble B in the cells. The B concentrations inyeast cells expressing BOR2 were about 30% to 35% ofthose in cells carrying the empty vector (Fig. 1), i.e. the Bconcentrations in cells expressing BOR2 were reducedto one-third of those of the vector control. This outcomeis similar to that obtained when performing the sameexperiments with BOR1 (Takano et al., 2002), suggest-ing that BOR2 also encodes a functional efflux-type Btransporter.

Figure 1. The B concentrations in yeast cells expressing BOR1 orBOR2. The concentrations of soluble B in yeast cells (mmol B kg–1 dryweight) are shown, given as the means 6 SD from three independenttransformants. DW, Dry weight.

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Cell-Type Specificity of BOR2 Expression

To examine the cell-type specificity of BOR2 expression,we generated transgenic Arabidopsis lines BOR2genome-GUS and BOR1genome-GUS that carry GUS fused togenomic fragments corresponding to the promoter andORF at the C termini (Fig. 2A). When the plants weregrown under low-B conditions, GUS activity derivedfrom BOR2-GUS was detected throughout the root,with staining in the meristematic and elongation zones,and in cortical cells in the mature portion of the roots(Fig. 2A). GUS activity derived from BOR1-GUS wasdetected in root meristematic and transition zones andaround vascular tissues during root cell maturation(Fig. 2A). To further clarify the cell-specific expressionof BOR1 and BOR2, we established transgenic Arabidopsisplants carrying both BOR2genome-GFP and BOR1genome-mCherry to coexpress BOR2-GFP and BOR1-mCherryfusion protein under the control of their own promoter(Fig. 2B). BOR2-GFP was predominantly observed inlateral root caps and epidermis of elongation zones.BOR2-GFP was also detected in all cells of the rootmeristematic zone, but little was observed in transitionzones. BOR1-mCherry was expressed in meristem andtransition zones and the epidermis and endodermis ofelongation zones but not in lateral root caps. This resultfor BOR1-mCherry is mostly consistent with previousreports of studies that employed BOR1-GFP (Takanoet al., 2010). These observations suggest that cell speci-ficity of BOR2 expression overlaps, in part, that of BOR1but also shows some differences.

Subcellular Localization of BOR2

In the transgenic lines carrying BOR2genome-GFP,GFP fluorescence was detected at the cell periphery(Fig. 2B). Visualization of the plasma membrane withFM4-64 revealed that BOR2-GFP colocalized with FM4-64staining and was not localized to the tonoplast (Fig. 3A).These observations demonstrate that BOR2 localizes tothe plasma membrane. Upon exposure to 400 mM man-nitol for 3 min to induce plasmolysis, BOR2-GFP was ob-served in the shrunken protoplasm (Fig. 3B). This showsthat BOR2 is nonstably localized to the plasmamembraneand is easily internalized to the cytoplasm.

BOR1 exhibits polar localization in the inner (inward)domain of the plasma membrane in all types of rootcells (Takano et al., 2010; Fig. 2B). As was the case withBOR1, BOR2-GFP shows polarity toward the inner plasmamembrane domain in the cells of the meristematic zone(Fig. 2B), although polar localization of BOR2-GFP wasweak in epidermis of the elongation zone (Fig. 2B).

To investigate the recycling of BOR2, we exposed thetransgenic BOR2genome-GFP plants to brefeldin A (BFA)in the presence of the protein synthesis inhibitor cy-cloheximide (CHX) under low-B conditions. BFA is aninhibitor of guanine-nucleotide exchange factors forADP-ribosylation factor, which inhibits exocytosis butnot endocytosis, and causes endosomal aggregation(Robinson et al., 2008). BOR2-GFP was found to be colo-calized in the BFA body with FM4-64, an endocytic tracer,in the epidermis of the elongation zone and lateral rootcaps (Fig. 3C), suggesting that BOR2-GFP cycles betweenthe plasma membrane and endosomes in a manner sim-ilar to BOR1 (Takano et al., 2005) under low-B conditions.

In the presence of high B concentrations, BOR1 isdegraded in the vacuole via the endocytic pathway(Takano et al., 2005). The transgenic plants carryingBOR2genome-GFP were transferred from the mediacontaining 0.3 mM boric acid to media containing either0.3 or 100 mM boric acid and incubated in darkness for150 min. GFP fluorescence was detected in the plasmamembrane in the presence of 0.3 mM B, but GFP wasobserved in vacuoles in medium containing 100 mM B(Fig. 3D). These results suggest that the accumulationof BOR2 is also regulated at the level of protein deg-radation in response to B conditions, similar to BOR1.

Growth of Loss-of-Function Mutants underLow-B Conditions

To examine the effects of BOR2 function loss on plantgrowth and B transport, we established transgenic Arabi-dopsis lines homozygous for T-DNA insertions in BOR1or BOR2 (Alonso et al., 2003; Rosso et al., 2003) and de-termined the sites of the insertions. The lines bor1-3(SALK_37312), bor2-1 (SALK_56473), and bor2-2 (GABI527H04) were all found to carry T-DNA insertions inthe exons of the corresponding genes (Fig. 4A; Kasaiet al., 2011). The growth of the T-DNA insertion mutantbor1-3was not significantly different from that of bor1-1,which carries a point mutation in the BOR1 ORF.

Figure 2. Cell specificity of BOR2 expression. A, GUS histochemicalstaining of roots of transgenic Arabidopsis roots carrying BOR2genome-GUS(BOR2g-GUS) and BOR1genome-GUS (BOR1g-GUS). Bars = 200 mm.B, GFP and mCherry imaging of a transgenic Arabidopsis root carryingboth BOR2genome-GFP (BOR2g-GFP) and BOR1genome-mCherry(BOR1g-mCherry). Bars = 100 mm. Plants were grown in solid mediacontaining 0.3 mM boric acid for 4 d.

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Boron Transport and Root Elongation



Wild-type ecotype Columbia (Col-0), bor1-3, bor2-1,bor2-2, and bor1-3/bor2-1 plants were grown on solidmedia containing between 0.1 and 3,000 mM boric acid.The growth of the bor2-1 and bor2-2 mutant plants wasless than that of wild-type plants on medium containingless than or equal to 1 mM boric acid (Fig. 4, B–D). Thegrowth patterns of bor2-1 and bor2-2were similar, stronglysuggesting that these defects are caused by the disruptionof BOR2.

The shoot fresh weights of bor2-1 and bor2-2were 26%and 25% (0.1 mM), 24% and 26% (0.3 mM), 34% and 34%(0.6 mM), and 58% and 69% (1 mM), respectively, of thoseof wild-type plants when the B supply was limited(Fig. 4C). In bor1-3 plants, shoot growth was severelyreduced to approximately 10% (0.1, 0.3, 0.6, and 1 mM)and 17% (3 mM) of those of the wild type under B-limitedconditions, as reported previously for bor1-1 (Noguchiet al., 1997; Takano et al., 2001). The extent of shootgrowth inhibition in bor2-1 and bor2-2was not as severeas that in bor1-3 (Fig. 4, B and C).

Root growth, by contrast, was more severely affectedin the bor2-1 and bor2-2 mutants than in the bor1-3mutants (Fig. 4B). The lengths of the primary roots inbor2-1 and bor2-2were reduced to 32% and 33% (0.1 mM),59% and 61% (0.3 mM), 61% and 65% (0.6 mM), and 78%and 77% (1 mM), respectively, of that in wild-type plants(Fig. 4D). These growth defects did not occur whenplants were grown under sufficient B concentrations(30 mM), suggesting that BOR2 functions mainly underlow-B conditions.

bor1-3/bor2-1 double mutant plants had much moresevere growth defects than any of the single insertionlines (Fig. 4, B–D). The fresh shoot weights of doublemutants grown under boric acid concentrations less thanor equal to 1 mM were not measured because of poorgrowth. The double mutant plants grew normally inthe presence of 30 mM boric acid. These results suggestthat BOR1 and BOR2 have major and at least partiallyoverlapping roles in shoot and root growth underB-limited conditions.

B Accumulation in the Aerial Portions of BOR2Mutant Plants

Because BOR1 is involved in xylem loading of B, wedetermined the amounts of B in the aerial portions ofwild-type and mutant plants grown hydroponically inthe presence of 0.1, 0.3, 3, 30, or 100 mM boric acid toexplore the functions of BOR2 in B translocation. Therewere no significant differences in the shoot dry weightsbetween BOR2 mutants and the wild type under allconditions tested (Fig. 5A). The shoots of bor2-1 andbor2-2 plants grown in medium containing less than orequal to 3 mM boric acid contained lower amounts of Bthan the shoots of wild-type plants, but there were nosignificant differences in the B contents in plants grown inmedium containing greater than or equal to 30 mM boricacid (Fig. 5B). However, in bor1-3 and bor1-3/bor2-1 dou-ble mutant plants, there were significant reductions in

Figure 3. Subcellular localization of BOR2 in the transgenic linescarrying BOR2genome-GFP (BOR2g-GFP). A, GFP imaging and FM4-64 staining of root epidermis of the transgenic Arabidopsis. Plants weregrown in solid media containing 0.3 mM boric acid for 9 d and treatedwith 4 mM FM4-64 for several minutes. Bars = 10 mm. B, GFP imagingof root epidermis exposed to 400 mM mannitol for 3 min for plas-molysis. GFP (left) and bright field (right) are shown. Plants were grownin solid media containing 1 mM boric acid for 5 d. Bars = 25 mm.C, GFP imaging of the transgenic root epidermis and root tip treatedwith BFA. Five-day-old plants grown under 0.3 mM boric acid wereexposed to 50 mM CHX for 30 min and 25 mM FM4-64 for 2 min,washed, and supplied with 50 mM CHX and 50 mM BFA for 30 min.GFP (left), FM4-64 (middle), and a merged image (right) are shown.Bars = 25 mm. D, GFP imaging of the transgenic root epidermistransferred to high B conditions. Plants were grown in solid mediacontaining 0.3 mM boric acid for 9 d and then transferred to the mediacontaining 0.3 or 100 mM boric acid. They were incubated for 150 minunder the dark condition. Bars = 25 mm.


Miwa et al.



the B contents in the aerial portions in media containing0.1, 0.3, 3, or 30 mM boric acid. Hence, BOR2 likelycontributes to the root-to-shoot translocation of B underlimited B supply conditions, but its contribution is muchsmaller than that of BOR1.

Reduction in Root Epidermal Cell Length in BOR2 MutantLines Grown under B-Limited Conditions

Because root growth was more affected in BOR2mutants than in the BOR1mutant in solid media underB-limiting conditions (Fig. 4, B and D), we observedroot cells after low B exposure to determine the natureof the root growth inhibition in BOR2 mutant plants.

Plants were first grown with 30 mM boric acid for 4 d andthen transferred to media containing 0.001 (wild-typeplants only), 0.1, or 30 mM boric acid. After 2 d, we exa-mined the epidermal cells in the root hair zones, whichdeveloped after the transfer (Fig. 6). In plants trans-ferred to medium containing 0.1 mM boric acid, theaxial lengths of the root epidermal cells in bor1-3, bor2-1,bor2-2, and bor1-3/bor2-1 were reduced compared withthose in the wild type. The double mutant had thegreatest reduction in cell length, followed in order bybor2-1 and bor2-2, with the least reduction in bor1-3. Inbor2-1, bor2-2, and bor1-3/bor2-1 plants transferred to me-dium containing 0.1 mM boric acid, the root epidermalcells were not only short but were also swollen and ir-regular in shape.

Figure 4. Characterization of T-DNA insertionmutants and their growth in media containingvarious concentrations of B. A, Schematic repre-sentation of T-DNA insertions in the BOR1 andBOR2 genes and the exon-intron structures of thegenes. Black boxes and bars indicate exons andintrons, respectively. The numbers shown at theright borders (RB) and the left borders (LB) cor-respond to the relative nucleotide positions fromthe first codon (+1) in the genomic sequences.The numbers in brackets are the nucleotide po-sitions in the cDNA sequences. B, Fourteen-day-old plants of the T-DNA insertion lines grown insolid media supplemented with 0.1 mM (left) and30 mM boric acid (right). A magnified view ofbor1-3/bor2-1 is shown at the lower right of theleft section. Bars = 10 mm. C, Fresh weights ofaerial portions of the T-DNA insertion lines col-lected from plants grown for 21 d on solid mediacontaining various concentrations of boric acid.Means 6 SD are shown (n = 3–12). D, Primaryroot lengths of the T-DNA insertion lines grownfor 14 d on solid media containing various con-centrations of boric acid. Means 6 SD are shown(n = 3–19). In C and D, different letters aboveeach bar indicate a significant difference amongthe lines grown in the same B concentration(P , 0.05, Tukey’s test).





Wild-type plants exposed to more severe B deple-tion conditions (0.001 mM boric acid) also had reducedroot cell lengths and swollen root cells. The impairedcell growth seen in wild-type plants grown in 0.001 mM

boric acid is similar to that seen in the double mutantsgrown in 0.1 mM boric acid. Thus, root cell elongation

was at least inhibited by the BOR2 mutation when theplants were transferred from B-sufficient to B-deficientmedium, similar to the severe effects of B deficiency onwild-type plants.

Reduced RG-II-B Dimer Formation in BOR2Mutant PlantsGrown in B-Limited Medium

One primary function of B is the cross linking ofthe pectic polysaccharide RG-II in cell walls (O’Neillet al., 2001). We first determined the relationship be-tween borate cross linking of RG-II and the rootgrowth of wild-type Arabidopsis plants under low-Bconditions (Fig. 7). When wild-type plants were grownin solid media containing 0.001, 0.1, 0.3, 1, or 30 mM

boric acid for 8 d, the primary root lengths were re-duced along with the decreased B concentrations inthe media and were positively correlated with theproportion of RG-II dimer. It is noted that approxi-mately 60% of RG-II in roots was cross linked whenroot elongation was severely inhibited in mediumcontaining 0.001 mM boric acid. The graph suggeststhat at least 50% cross linking of RG-II is required tomaintain root growth.

Then, we determined the B concentrations and for-mation of RG-II cross links in roots to investigate thecauses of the growth defects observed in BOR2 T-DNAmutant plants (Table I). There were no significant differ-ences in the concentrations of total B in the roots of bor2-1,bor2-2, and wild-type plants; however, the relativeproportions of the RG-II-B dimer to the total RG-II inbor2-1 and bor2-2were reduced to 45%, whereas that ofwild-type plants was reduced to 54% when plants weregrown on medium supplemented with 0.1 mM boricacid. At 0.1 mM boric acid, the B concentrations of theroots of bor1-3 and bor1-3/bor2-1 were 15% and 20%lower than in wild-type plants, respectively. The rateof formation of the RG-II-B dimer in bor1-3 roots wasnot significantly different from that in wild-type plants.

Figure 5. B concentrations in aerial portions of BOR2 mutant plants.Col-0, bor1-3, bor2-1, bor2-2, and bor1-3/bor2-1 plants were grown hy-droponically for 30 d in media containing 0.1, 0.3, 3, 30, or 100 mM boricacid. The dry weights (A) and the B concentrations (B) of the aerial portionsof the plants were determined. Means 6 SD are shown (n = 3–5). Differentletters above each bar indicate a significant difference among lines re-ceiving the same B treatment (P , 0.05, Tukey’s test). DW, Dry weight.

Figure 6. Root cell elongation in mu-tant lines in response to changes in theB concentration of the medium. Plantswere grown for 4 d in solid mediacontaining 30 mM boric acid, trans-ferred to medium supplemented with0.001, 0.1, or 30 mM boric acid, andincubated for 2 d. Epidermal cells ofroot hair zone that developed after themedium transfer were observed. Bars =100 mm.


Miwa et al.



In bor1-3/bor2-1, the relative proportions of the RG-II-Bdimer were reduced to levels similar to those in bor2-1and bor2-2. All lines had greater than 90% RG-II-B di-mer formation when grown on medium supplementedwith 30 mM boric acid. In summary, the BOR2 mutationdid not affect the concentrations of total B in roots butinfluenced the formation of RG-II-B dimers in rootsexposed to limited B supply. Assuming that a reductionof even 10% in RG-II cross linking significantly affectsroot growth (Fig. 7), the reduction in RG-II-B dimer for-mation from 54% to 45% (Table I) is likely to be a majorcause of the reduced cell elongation seen in T-DNAmutant plants of BOR2 grown under low-B conditions.

DISCUSSION

BOR2 Is Essential for Root Growth underLow-B Conditions

BOR2 was characterized as an efflux-type B trans-porter localized to the plasma membrane, which is alsotrue for BOR1. However, the major physiological roles

of BOR1 and BOR2 in plants are different: BOR1 func-tions in xylem loading of B, whereas BOR2 functions inroot cell elongation under B-limited conditions.

The shoot B concentrations were lower in BOR2mutant plants than in wild-type plants, although thereduction was greater in bor1-3 under low-B conditions(Fig. 5B). Hence, BOR2 also functions in the transportof B to shoots, but the extent of this contribution, if any,is minor compared with that of BOR1. The predominantexpression of BOR1 in the endodermis and its polarlocalization in the inner plasma membrane domain(Takano et al., 2010; Fig. 2B) are likely to be major fac-tors in B transport into the stele, which is a prerequisitefor subsequent transport of B from the root to the shootvia the xylem.

It is interesting to note that BOR1 and BOR2 are bothefflux-type B transporters exhibiting polar localizationin plasma membrane and internalization and that tissuespecificity of their expressions are in part overlapping toeach other (Figs. 2 and 3), while their major physio-logical roles are different: BOR1 for xylem loading andBOR2 for cross linking of pectins (this study). Althoughwe cannot rule out the possibility that there may be adifference in transport properties and/or differentialregulation of proteins, given the evidence, it is reason-able to surmise that the major reason for the differencesin physiological roles between BOR1 and BOR2 are thedifferential tissue-specific patterns.

In addition to its minor contribution to the root-to-shoot translocation of B, BOR2 was found to be requiredfor normal root growth (Fig. 4, B and D), particularly forroot cell elongation (Fig. 6), under conditions of limitedB supply. The expression of BOR2 in the meristematiczone and the epidermis of the elongation zone (Fig. 2)is likely to support root cell elongation. The high-B-induced degradation of BOR2 (Fig. 3D) is consistentwith the importance of BOR2 under low-B conditions.

Borate Cross Linking of RG-II Is Important forRoot Growth

One of the physiological roles of B in shoots is thecross linking of RG-II chains in the cell wall (O’Neill

Figure 7. Relation between primary root lengths and proportion of RG-IIdimer. Col-0 plants were grown in solid media containing 0.001, 0.1, 0.3,1, and 30 mM boric acid for 8 d. Cell wall fractions were isolated, and therelative proportions of RG-II-B dimer to total RG-II were determined. Rel-ative proportion of RG-II-B (%) is shown in a longitudinal axis as a logscale, and an approximation straight line is presented. Mean 6 SD areshown (n = 52–239 for primary root length, n = 3–4 for RG-II measure-ment). Primary root lengths were reduced along with the decreased Bconcentrations in the media, and each dot corresponds to the data obtainedfrom plants grown under 0.001, 0.1, 0.3, 1, and 30 mM boric acid supply.

Table I. RG-II-B dimer formation and B concentration of Arabidopsis roots grown under low-B and normal-B conditions

Plants were grown on solid media containing 0.1 and 30 mM boric acid for 10 to 14 d. Cell wall fraction was prepared from plant roots and thentreated with EPG. The solution of EPG-soluble material was fractionated by size-exclusion chromatography equipped with a refractive index detector.Relative proportions of RG-II-B dimer to total RG-II and B concentration of whole roots are shown (means6 SD, n = 3–4). Different superscript lettersshow significant difference (P , 0.05, Tukey’s test).

Plant line

B Concentration in Roots Relative Proportion of RG-II-B Dimer

0.1 mM B Concentration in

Medium

30 mM B Concentration in

Medium

0.1 mM B Concentration in

Medium

30 mM B Concentration in

Medium

mmol kg–1 dry wt %

Col-0 0.39 6 0.03a 1.44 6 0.17 54.0 6 1.1a 90.1 6 4.1bor1-3 0.33 6 0.01ab 1.54 6 0.04 52.8 6 0.4a 90.0 6 1.1bor2-1 0.36 6 0.04ab 1.28 6 0.15 42.5 6 0.5b 91.6 6 2.1bor2-2 0.37 6 0.02ab 1.53 6 0.05 45.7 6 1.0b 92.0 6 2.3bor1-3/bor2-1 0.31 6 0.03b 1.43 6 0.12 45.1 6 0.9b 90.4 6 1.5





et al., 2001). Although this role has yet to be demon-strated definitively in roots, the preferential distribu-tion of B in root cell walls and the presence of RG-II-Bsuggest that B also functions in the cross linking ofRG-II molecules in roots. B is distributed primarily tocell walls prior to its accumulation in the soluble fractionof Arabidopsis roots under low-B conditions (Noguchiet al., 2000). The RG-II-B complex is known to occur inradish roots (Kobayashi et al., 1996) and pumpkin(Cucurbita maxima) roots (Matsunaga and Ishii, 2006).

In a study of pumpkin roots exposed to low-B con-ditions, RG-II-B dimer formation decreased along withthe root dry weight (Matsunaga and Ishii, 2006). Inpumpkin roots subjected to B-depleted conditions (noaddition of B), the fraction of RG-II forming the boratecomplex was about 55% (Matsunaga and Ishii, 2006).Under these conditions, the pumpkin root dry weightwas about 50% lower than under the B-sufficient con-ditions. A positive correlation was also found betweenroot growth and the proportion of RG-II-B formationunder a wide range of B-deficient conditions in ourstudy of wild-type Arabidopsis plants (Fig. 7). The pro-portion of RG-II cross linking reported for pumpkinroots is comparable to our present data for wild-typeArabidopsis roots (Fig. 7; Table I). These observationsalso support the idea that the occurrence of RG-II-B is adeterminant of root growth, especially root cell elonga-tion, as B depletion inhibits root cell elongation (Fig. 6).Furthermore, our study suggests that 50% RG-II crosslinking is likely to be a lower limit to support plant rootelongation.

The total root B concentration was not significantlydifferent in bor2-1 and bor2-2 under limited B supply,but the fraction of RG-II forming the borate complex inbor2-1 and bor2-2 was reduced to 43% to 46%, whereasthat of the wild type under the same B condition was54% (Table I). It is reasonable to assume that the further

reduction of borate-cross-linked RG-II caused by themutation in BOR2 under B-deficient conditions wasdetrimental to cell elongation and resulted in reducedroot elongation (Figs. 4 and 6). Given all of these ob-servations, we propose that decreased RG-II-B dimerformation is the major inhibitor of root cell elongationin BOR2 mutants grown in limited B.

The Role of BOR2 in B Transport for Efficient RG-II-BComplex Formation: A Model

Immunocytochemical analysis in radish roots usingantibodies against RG-II suggested that RG-II is pre-sent in all root tip cells and that it is located extremelyclose to the plasma membrane beneath the cell wall(Matoh et al., 1998; Matoh, 2001). RG-II-B dimers formspontaneously in vitro, and the reaction is stimulatedby the addition of divalent cations (O’Neill et al., 1996;Ishii et al., 1999). Under physiological conditions, B ispresent mainly as boric acid in solution; boric acid isa Lewis acid that interacts with water molecules toform borate anion (B[OH]3 + H2O = B[OH]4

– + H+;pKa = 9.24; Power and Woods, 1997). Borate anionforms a cis-diol ester with the apiosyl residues in RG-II(Ishii and Ono, 1999) and is thus likely preferred overboric acid as the substrate for RG-II dimer formation.However, molecular mechanisms for borate RG-II di-merization are still largely unknown, i.e. the site andthe substrates of the reaction in vivo.

Root cell elongation is also severely reduced in loss-of-function mutants of NIP5;1, a boric acid channel forB uptake that is required for growth under B-limitedconditions (Takano et al., 2006). GFP-NIP5;1 expressedunder the control of its own promoter was detected inthe root cap and epidermal cells in the elongation zoneof the roots (Takano et al., 2010). These findings suggest

Figure 8. Schematic model of the roles of BOR2in root cells. A possible role for BOR2 in rootmeristems under conditions of low-B supply isshown. One hypothesis is that boric acid/borate isexported out of the cell by BOR2 to the apoplast,where it is used in the efficient cross linking ofRG-II molecules. Another possibility is that BOR2concentrates boric acid/borate into secretionvesicles to facilitate dimerization of RG-II in thevesicles, and then the dimeric RG-II is secreted tothe apoplast. The thickness of the arrows signifiesthe efficiency of the reaction. mRG-II, RG-IImonomer; dRG-II-B, RG-II-B dimer.


Miwa et al.



that B transport via the apoplast and simple diffusionacross the plasma membrane are not able to satisfythe demand for B during root cell elongation underB-limited conditions. Because BOR2 is expected to ex-port B from the cytosol, it is reasonable to propose thatunder conditions of limited B supply, the B requiredfor root cell elongation comes mainly from the cytosolthrough BOR2.A model for BOR2 function is presented in Figure 8.

In medium containing low B concentrations, the B con-centrations seem insufficient for RG-II-B dimer formationin apoplast. One model is that a portion of the B enteringcells through NIP5;1 or by passive diffusion and theboric acid/borate is supplied by the BOR2 exporter tothe RG-II monomer in apoplast, resulting in efficientdimer formation. RG-II is likely present near the plasmamembrane; thus, a B exporter located in this membranewould be able to directly control RG-II cross linking. Itis also reasonable to assume that there is a specific in-teraction, either directly or indirectly, between BOR2and a protein, if any, that facilitates borate cross linkingto establish the efficient transport of boric acid/borateinto RG-II.It is also possible that RG-II-B forms in the secretion

vesicles. We demonstrated that BOR2-GFP was localizedto endosomal aggregations following BFA treatmentunder B-limited conditions (Fig. 3C), suggesting that asignificant quantity of BOR2 is located in the secretory/recycling pathway between the plasma membrane andtrans-Golgi network. It is possible that BOR2 localizedto the secretion vesicles transports boric acid/borate intothem, resulting in increased B concentrations in thesecompartments and facilitating RG-II-B dimer formation.In conclusion, we demonstrated that BOR2 is in-

dispensable for root growth and RG-II-B cross linkingin cell walls under B-limited conditions. Our findingsare an example of the involvement of mineral trans-porters in cell wall formation and maintenance duringnormal plant growth.

MATERIALS AND METHODS

Plant Culture and Transformation

Arabidopsis (Arabidopsis thaliana) wild-type (Col-0) was from our laboratorystock. Plants were grown on solid media (Fujiwara et al., 1992) in which the Bconcentrations were adjusted with boric acid. To reduce B contamination, ul-trapure water by Milli-Q Synthesis A-10 (Merck Millipore) and autoclavablepolypropylene bottles were used. For growth tests, the solid media contained2% (w/v) Suc and 1.5% (w/v) gellan gum (Wako Pure Chemicals), and for RG-IIdimer formation in the wild type, the media contained 1% (w/v) Suc and1% (w/v) gellan gum. Surface-sterilized seeds were sown on the solid mediaand incubated for two days at 4°C. The plates were then placed vertically andincubated at 22°C under a 16-h-light /8-h-dark cycle. For the determination of Bamounts in shoots, Arabidopsis plants were grown hydroponically as described(Takano et al., 2001). To generate transgenic Arabidopsis, plasmids were intro-duced into Agrobacterium tumefaciens strain GV3101 (C58C1Rifr) pMP90 (Gmr)and transformation was performed by the floral dip method. For selection oftransformants, solid media that contain one-half strength of Murashige Skoogsalt, 1% (w/v) Suc and 0.2% (w/v) gellan gum, and hygromycin (20 mg L–1) wasused. For selection of plants expressing GFP or mCherry-tagged proteins, T1plants resistant to hygromycin were transferred to solid media (Takano et al.,2005) containing 0.3 mM boric acid, and fluorescence-positive plants were se-lected under a macrozoom fluorescent microscope (MVX10; Olympus).

Cloning of Full-Length cDNA

Full-length cDNAs for all of six paralogs were obtained, and DNA sequencesof the clones were determined. The cDNAs for At1g15460, At3g06450, andAt3g62270 were obtained from the RIKEN Genomic Sciences Center, and thosefor At1g74810, At4g32510, and At5g25430 were isolated by reverse transcription-mediated PCR. cDNA of At1g74810 was obtained from Arabidopsis roots, andthose of At4g32510 and At5g25340 were obtained from flowers.

Generation of Yeast Expressing BOR2 and Determinationof B Concentrations in Yeast Cells

BOR2 cDNAORF was amplified using KOD Plus DNA polymerase (TOYOBO)with the primer set (59-TGAGAGGTACCAGAGCCATGGAAGAGACTT-TTGTTCCGTTTGA-39 and 59-GTTTTAGCATGCTCATTTCGACGATGAT-GATGGGT-39). The PCR products were digested with KpnI and SphI andinserted into the corresponding sites in pYES2, resulting in pTF524 (BOR2coding sequence in pYES2). The plasmids pTF477 (BOR1), pTF524 (BOR2), andpYES2 were introduced into yeast (Saccharomyces cerevisiae) strain DScBOR1(Winzeler et al., 1999). Transformants were selected on synthetic minimal medium(Sherman, 1991) supplemented with 2% (w/v) Glc, 20 mg L–1 His, 30 mg L–1 Leu,and 20 mg L–1 Met. The yeast medium used in this experiment was syntheticminimum medium containing 2% (w/v) Gal. Fifty milliliters of culture at themidlog phase were transferred to a 50-mL plastic tube and centrifuged at3,000g for 5 min to collect the yeast cells. The cells were mixed with 20 mLmedium (adjusted to pH 5.5 with Tris) containing 0.1 or 0.5 mM boric acid andincubated for 60 min. Sampling of the soluble fraction in the yeast cells anddetermination of the B concentrations were conducted as described by Takanoet al. (2002). Experiments were performed with three independent transformants.

T-DNA Insertion Mutants and Determination of T-DNAInsertion Sites

T2 seeds of bor1-3 (SALK_37312), bor2-1 (SALK_56473), and bor2-2 (GABI527H04) were provided by the Salk Institute (Alonso et al., 2003) and the MaxPlanck Institute (Rosso et al., 2003). From the T2 plants provided, homozygouslines were selected by PCR and the flanking sequences were determined bysequencing. All of the plant lines were in the Col-0 background. The primersused for bor1-3 and bor2-1were described in Kasai et al. (2011). The primers forbor2-2, 59-ATTGCCAATTGAAGTCAAAG-39 (forward) and 59-TTTTACC-GACATTTGACATGGTA-39 (reverse) and the GABI left border, 59-ATATT-GACCATCATACTCATTGC-39, were used.

Generation of Transgenic Plants Expressing GUS andFluorescence Protein Fusions under the Control ofOwn Promoter

For generation of BOR2 construct, the genomic nucleotides 23053503 to23059413 of chromosome 3 sequences corresponding to putative promoterregion (2.8 kb) and ORF (3.1 kb) was amplified by PCR using primers59-CACCTCTAGATGAATAATTAATAT-39 and 59-TTTCGACGATGATGATG-GGTTTAA-39. The fragment was cloned into pENTR/D-TOPO (Invitrogen)and then into pMDC163 (Curtis and Grossniklaus, 2003) and pGWB504(Nakagawa et al., 2007a) for GUS and GFP fusion by LR reaction in Gatewaytechnology (Invitrogen), respectively. The resulting plasmids were named aspKM20 for BOR2genome-GUS and pST1 for BOR2genome-GFP. mCherryfragment was amplified by PCR with the primers 59-CGCCTCGACTCTA-GAATGGTGAGCAAGGGCGAGGA-39 and 59-GGGAAATTCGAGCTCCT-ACTTGTACAGCTCGTCCA-39 using wave1R (Geldner et al., 2009) as atemplate. The fragment was cloned into the XbaI and SacI sites of pMDC163by a In-Fusion HD cloning kit (Takara). The resulting Gateway destinationvector containing mCherry was named as pKI1. For the BOR1 construct, thegenomic sequence corresponding to promoter and ORF was amplified withthe primers 59-AAAAAGCAGGCTCGGGAGATGACTAACAACGACAC-39and 59-AGAAAGCTGGGTGTTCGATGACGACTGGTTCAAGG-39. The DNAfragment was cloned into pDONR/zeo by BP reaction and then into pKI1and pMDC164 (Curtis and Grossniklaus, 2003) for mCherry and GUS fusionby LR reaction with Gateway technology (Invitrogen), resulting in pKI2 forBOR1genome-mCherry and BOR1-pMDC164 for BOR1genome-GUS, respectively.pKM20 and pST1 were introduced into bor2-1, and BOR1-pMDC164 was in-troduced into bor1-1. bor1-3/bor2-1were cotransformed with pST1 and pKI2. GFP





fluorescence in the transgenic lines carrying BOR2genome-GFP (pST1) wassimilar to the cotransformed lines carrying both BOR2genome-GFP (pST1) andBOR1genome-mCherry (pKI2; Supplemental Fig. S1A; Fig. 2B). Introduction of theconstruct BOR2genome-GFP (pST1) into bor2-1 restored the bor2-1 growth under0.3 mM boric acid in T2 generation, suggesting that BOR2-GFP was func-tional (Supplemental Fig. S1B).

GUS Staining

GUS staining was carried out with transgenic plants in T2 generation forBOR2genome-GUS and in T3 for BOR1genome-GUS. Plants were grown for4 d in medium containing 0.3 mM boric acid. GUS staining was conducted asdescribed in Shibagaki et al. (2002). Optical images were taken with a lightmicroscope (BX50WI; Olympus).

GFP Imaging

Laser scanning confocal microscopy was performed using TCS-SP8 equippedwith a water-immersed 340 lens (Leica Microsystems). For imaging of GFP andFM4-64 (Molecular Probes), excitation wavelength was 488 nm and the detectionwavelengths were 500 to 540 nm for GFP and greater than 640 nm for FM4-64.Dual-color images of GFP and mCherry were acquired by sequential lineswitching, allowing the separation of channels by both excitation and emissionwith the following excitation and detection wavelengths: 488 nm and 505 to 530for GFP and 552 nm and 600 to 700 for mCherry. The plants were grown onvertically placed solid medium (Takano et al., 2005) containing 0.3 or 1 mM

boric acid, 2% (w/v) Suc, and 1.5% (w/v) gellan gum (Wako Pure Chemicals) for4 to 9 d. As an endocytic tracer, a styryl dye FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide] was used.FM4-64 was prepared as 10 mM stock solution in water and used at 4 mM,except for the experiments using BFA (Sigma). For plasmolysis, plants wereexposed to 400 mM mannitol for 3 min. Stock solution of CHX (Sigma) andBFA were prepared at 50 mM in water and 50 mM in dimethyl sulfoxide, re-spectively. For BFA treatment, plants were grown in solid media containing1 mM boric acid for 5 d. Then, knife-cut root tips (10 mm) were incubated with50 mM CHX for 30 min and then 25 mM FM4-64 for 2 min in a plastic tube witha lid open. After washing the roots, they were treated with 50 mM CHX and50 mM BFA for 30 min. To expose high-B concentrations, plants were firstgrown in solid media containing 0.3 mM boric acid for 9 d. Then, the plantswere transferred to the media containing 0.3 or 100 mM boric acid and incu-bated in darkness for 150 min. Solutions for each treatment were prepared inthe liquid medium (Takano et al., 2005).

Observation of Root Cells under Low-B Conditions

Plants were grown for 4 d in solid media containing 30 mM boric acid andtransferred to medium supplemented with 0.001 (only for Col-0), 0.1, or 30 mM boricacid. Positions of primary root tip at 24 h after the medium transfer were marked.Two days after the medium transfer, epidermal cells located at the markedportion were observed. Plant roots were cut and treated with 10 mg mL–1

propidium iodide (Molecular Probes). They were observed using a confocallaser microscopy Zeiss LSM 510 with a 543-nm excitation wavelength and anemission window of greater than 560 nm for propidium iodide.

Analysis of B Concentrations in Shoots and Roots

For B measurements of shoots, plants were grown hydroponically in mediasupplemented with 0.1, 0.3, 3, 30, or 100 mM boric acid (Takano et al., 2001).Aerial portions of 30-d-old hydroponically cultured plants were harvested. Forroots, plants grown on solid media containing 0.1 or 30 mM boric acid for 10 to14 d were harvested. The plant tissues were dried at 60°C for more than 48 h,and then the dry weight was measured. The tissues were put in 8-mL Teflontubes and digested at 130°C for 3 h with 1 mL of concentrated HNO3. The acid-digested samples were dissolved with 3 mL of the solution containing 5 mg L21

beryllium in 0.08 N HNO3 and diluted with the same solution as appropriate.The B concentrations were then determined by inductively coupled plasma massspectrometry (model SPQ9000; SII).

Determination of RG-II-B Dimer Ratio

To determine RG-II dimer formation in wild-type plants, plants weregrown in solid media containing 0.001, 0.1, 0.3, 1, and 30 mM boric acid for 8 d.

For one sample for cell wall extraction, 80 plants (0.001 and 0.1 mM) and 40plants (0.3, 1, and 30 mM) were used. For comparison of RG-II dimerizationamong mutant lines, plants were grown on solid media containing 0.1 or30 mM boric acid for 10 to 14 d. To prepare a cell wall fraction, roots wereharvested and homogenized in 80% (v/v) ethanol. Insoluble residues aftercentrifugation were washed with 80% (v/v) ethanol twice, 99.5% (v/v) ethanol,chloroform/methanol twice, acetone, and water twice. Alcohol-insoluble resi-dues considered as cell wall materials were treated with 0.1 M NaOH for morethan 4 h at 4°C to saponify methyl and acetyl esters and then adjusted to pH 5.0with 10% (v/v) acetic acid. They were digested with endopolygalacturonase(EPG) for 24 h at 4°C or 16 h at 35°C. We have already confirmed that EPGtreatment at 4°C did not make differences from that at 35°C in the case ofArabidopsis wild-type roots. After centrifugation, the supernatant was filteredthrough a 0.2- or 0.45-mm membrane. They were subjected to size-exclusionHPLC/refractive index detector (Hitachi High Technologies Corporation) andanalyzed as described in Matsunaga and Ishii (2006). The relative proportionsof RG-II-B dimer to total RG-II were estimated from the peak area corre-sponding to RG-II-B dimer to the integration of the respective peak area ofRG-II monomer and RG-II-B dimer.

Sequence data from this article can be found in the GenBank/EMBL data-bases under accession numbers GU971377 (BOR6, At5g25430) and GU971378(BOR7, At4g32510).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Functional complementation of Arabidopsis car-rying BOR2genome-GFP (BOR2g-GFP).

ACKNOWLEDGMENTS

We thank Tsuyoshi Nakagawa and Niko Geldner for providing vectors,the technical assistance of Yuko Kawara and Kayoko Aizawa, Japanese Societyfor the Promotion of Science Research Fellowships for Young Scientists, andHokkaido University Leader Development System in the Basic InterdisciplinaryResearch Areas.

Received July 31, 2013; accepted October 9, 2013; published October 10, 2013.

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Roles of BOR2, a Boron Exporter, in Cross Linking of … · BOR2 mutants were not signiﬁcantly different from those in wild-type plants, but the proportion of cross-linked RG-II