The Transcription Factor NIN-LIKE PROTEIN7 Controls Border-Like

The Transcription Factor NIN-LIKE PROTEIN7 ControlsBorder-Like Cell Release1[OPEN]

Rucha Karve, Frank Suárez-Román, and Anjali S. Iyer-Pascuzzi*

Purdue University, Department of Botany and Plant Pathology, West Lafayette, Indiana 47907

ORCID IDs: 0000-0002-9638-877X (F.S.-R.); 0000-0001-5356-4350 (A.S.I.-P.).

The root cap covers the tip of the root and functions to protect the root from environmental stress. Cells in the last layer of theroot cap are known as border cells, or border-like cells (BLCs) in Arabidopsis (Arabidopsis thaliana). These cells separate fromthe rest of the root cap and are released from its edge as a layer of living cells. BLC release is developmentally regulated, but themechanism is largely unknown. Here, we show that the transcription factor NIN-LIKE PROTEIN7 (NLP7) is required for theproper release of BLCs in Arabidopsis. Mutations in NLP7 lead to BLCs that are released as single cells instead of an entire layer.NLP7 is highly expressed in BLCs and is activated by exposure to low pH, a condition that causes BLCs to be released as singlecells. Mutations in NLP7 lead to decreased levels of cellulose and pectin. Cell wall-loosening enzymes such as CELLULASE5(CEL5) and a pectin lyase-like gene, as well as the root cap regulators SOMBRERO and BEARSKIN1/2, are activated in nlp7-1seedlings. Double mutant analysis revealed that the nlp7-1 phenotype depends on the expression level of CEL5. Mutations inNLP7 lead to an increase in susceptibility to a root-infecting fungal pathogen. Together, these data suggest that NLP7 controls therelease of BLCs by acting through the cell wall-loosening enzyme CEL5.

The root cap surrounds the root tips of nearly allangiosperms, gymnosperms, and pteridophytes (Barlow,2003) and is fundamental to root-environment interac-tions. This tissue is critical to plant health as it protects thedelicate root meristem (Barlow, 2003), senses gravity(Blancaflor et al., 1998, 1999) and water (Eapen et al.,2005), and secretes antimicrobial compounds and sec-ondary metabolites that defend the root against soil-borne pathogens and abiotic stress (Hawes et al., 1998,2000; Vicré et al., 2005; Driouich et al., 2013;Watson et al.,2015). The root cap is composed of two cell types thatsurround the meristem: the lateral root cap, which flanksthe meristem, and the columella root cap located at theroot tip (Fig. 1).

In Arabidopsis (Arabidopsis thaliana), the columellais composed of five to six layers of rectangular cells(Benfey and Scheres, 2000; Fig. 1). New columella cellsare produced from columella stem cells, and as they agethey progressively differentiate from amyloplast-filledcells that sense gravity to mucilage-secreting cells thatseparate from the rest of the root cap and are releasedfrom its edges (Vicré et al., 2005; Durand et al., 2009;

Bennett et al., 2010, 2014; Driouich et al., 2013). InArabidopsis, cells of the columella root cap are releasedas a layer of living cells that are attached to each other aswell as to one or two lateral root cap cells (Fig. 1; Vicréet al., 2005; Durand et al., 2009; Kumpf and Nowack,2015). Together, the columella and lateral root cap cellsthat are released in this last layer are called border-likecells (BLCs; Vicré et al., 2005; Kumpf and Nowack,2015). Despite their simultaneous release and physicalattachment, the mechanism of release for columellaBLCs and lateral root cap BLCs is very different. BLCsof the lateral root cap transition to programmed celldeath at the time of their release (Fendrych et al., 2014).In contrast, BLCs of the columella do not undergoprogrammed cell death (Fendrych et al., 2014; Kumpfand Nowack, 2015) and remain alive for at least 72 hafter release (Plancot et al., 2013). How columella BLCrelease occurs is not well understood.

Border cells and BLCs secrete root exudates includ-ing antimicrobial compounds and are an importantpart of the root’s defense against both biotic and abioticrhizosphere stresses (Hawes et al., 1998, 2000; Vicréet al., 2005; Driouich et al., 2013; Plancot et al., 2013;Watson et al., 2015). Both border cell and BLC releaserequire cell wall-degrading enzymes (Hawes and Lin,1990; Wen et al., 1999; Vicré et al., 2005; Durand et al.,2009). In Arabidopsis, BLC release occurs through theaction of enzymes that modify components of the cellwall, including cellulases and pectin methylesterases(Bouton et al., 2002; del Campillo et al., 2004; Durandet al., 2009). Mutants deficient in cell wall homo-galacturonan (HG), a major component of pectin (Wolfet al., 2009), release BLCs as single cells instead of anintact layer (Bouton et al., 2002; Durand et al., 2009).BLC release also depends on the proper degradation of

1This work was funded by Purdue University.* 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:Anjali S. Iyer-Pascuzzi ([email protected]).

R.K. performedmost of the experiments; F.S.-R. provided technicalassistance; R.K. and A.S.I.-P. designed the experiments and analyzedthe data; A.S.I.-P. supervised the experiments and wrote the articlewith input from all authors.

[OPEN] Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.16.00453

Plant Physiology�, July 2016, Vol. 171, pp. 2101–2111, www.plantphysiol.org � 2016 American Society of Plant Biologists. All Rights Reserved. 2101 www.plantphysiol.orgon February 14, 2018 - Published by Downloaded from

Copyright © 2016 American Society of Plant Biologists. All rights reserved.

http://orcid.org/0000-0002-9638-877X

http://orcid.org/0000-0001-5356-4350

mailto:[email protected]

http://www.plantphysiol.org

mailto:[email protected]

http://www.plantphysiol.org/cgi/doi/10.1104/pp.16.00453


cellulose, as a mutant defective in CELLULASE5 (CEL5)has a sticky root cap, with extra root cap cells stuck tothe root tip (del Campillo et al., 2004). Furthermore, atriple mutant defective in the transcription factorsSOMBRERO (SMB), BEARSKIN1 (BRN1), and BRN2has an extremely sticky root cap with masses of cellsremaining attached and has reduced CEL5 expression(Bennett et al., 2010). Although cell wall-loosening en-zymes are known to be necessary for BLC release, howtheir expression is controlled to ensure release of anintact layer of BLCs is not clear.

Here, we show that the transcription factor NIN-LIKE PROTEIN7 (NLP7) is required for the release of anintact layer of BLCs in Arabidopsis. Low pH stresscauses the release of BLCs as single cells from the roottip of wild-type plants, and a mutation in NLP7 sig-nificantly enhances this single cell release in bothstandard pH (pH 5.7) and low pH (pH 4.0) conditions.NLP7 encodes an Arabidopsis homolog of the noduleinception (NIN) transcription factor from Lotus japon-icas and has been previously described in Arabidopsisfor its role in nitrate signaling (Castaings et al., 2009;Marchive et al., 2013). NLP7 expression is activated bylow pH conditions and is highly expressed in BLCs. Theroot of the nlp7 mutant shows a decrease in celluloseand pectin content. Gene expression of SMB andBRN1/2 is activated in the mutant, as is the expressionof several cell wall-loosening enzymes, such as CEL5,

XYLOGLUCAN ENDOTRANSGLUCOSYLASE (XTH5),and a PECTIN LYASE-like gene (PL). Double mutantanalysis reveals that the nlp7 phenotype depends on theexpression level of CEL5. Consistent with a role for BLCsin biotic stress, mutations in NLP7 lead to increasedsusceptibility to the soil-borne fungus Fusarium oxy-sporum f. sp. conglutinans (Foc). Together, our data showthat NLP7 expression maintains pectin and celluloselevels in the root and represses the expression of CEL5,XTH5, PL, SMB, and BRN1/2, thereby preventing singlecell border-like cell release.

RESULTS

Low pH Stress Promotes Single-Cell BLC Releasein Arabidopsis

We previously found that among five different celltypes in the Arabidopsis root, low pH conditions mostsignificantly affected gene expression in the columellaroot cap (Iyer-Pascuzzi et al., 2011). Given that BCs areimportant for tolerance to Al toxicity (Li et al., 2000), amajor problem in acid soils (Kochian et al., 2015), wereasoned that BLCs may be affected by low pH condi-tions. Under standard conditions (pH 5.7), at 7 d afterimbibition (dai) over 85% of wild-type Arabidopsis rootsrelease BLCs as intact layers of physically attached cells,and,15% of wild-type roots release BLCs as single cells(Fig. 2, A and G). In contrast, examination of BLCs inplants grown under low pH stress (pH 4.0) showed thatonly 45% of wild-type roots release BLCs as intact layers,while approximately 55% of roots release BLCs as singlecells (Fig. 2, B and G). Low pH stress therefore promotesthe release of BLCs as single cells in Arabidopsis.

NLP7 Is Required for Columella Border-LikeCell Adhesion

To search for genes that may play a role in BLC re-lease, we examined a time course of gene expression inwhole roots after exposure to low pH (Iyer-Pascuzziet al., 2011). We reasoned that genes coexpressed withknown regulators of the low pH response may befundamental to low pH-induced phenotypes. K-meansclustering of 1848 genes differentially expressed through-out the time course (Iyer-Pascuzzi et al., 2011) identi-fied one cluster that contained the transcription factor(TF) Sensitive to Proton Rhizotoxicity (STOP1). Genesin this cluster were activated 6 to 12 h after exposure tolow pH stress (Supplemental Fig. S1). Mutations inSTOP1 lead to hypersensitivity to acidic pH and severeroot growth inhibition under low pH (Iuchi et al., 2007).We hypothesized that the 10 other TFs in this clustermay also play a role in the lowpH response. Becausewewere specifically interested in the BLC phenotype, weexamined the root cap in mutant lines for each of these10 TFs.

One of these lines (SALK_026134), with a T-DNAinsertion inNLP7, showed defective BLC release (Fig. 2,

Figure 1. The Arabidopsis root cap. Border-like cells are the last layer atthe tip of the root (arrow) and contain cells derived from both columellaand lateral root caps. Stars show the living columella border-like cells.White dashed line illustrates where border-like cell separation occurs torelease an intact layer of cells.

2102 Plant Physiol. Vol. 171, 2016

Karve et al.

www.plantphysiol.orgon February 14, 2018 - Published by Downloaded from Copyright © 2016 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org/cgi/content/full/pp.16.00453/DC1


C, D, and G). Quantitative RT-PCR (qRT-PCR) con-firmed thatNLP7was activated by lowpH (SupplementalFig. S2). Examination of the BLCs in SALK_026134 (here-after nlp7-1) seedlings showed that under standard pH,approximately 44% of roots released BLCs as single cells,compared to just 12% inwild-type plants and 19% in rootsof the complemented line pNLP7:NLP7:GFP/nlp7-1 (Fig.2G). Under low pH conditions, which loosen cells inthe root cap and promote single cell BLC release, over75% of nlp7-1 mutant roots released BLC as singlecells, compared to 55% of wild-type plants and 42% ofpNLP7:NLP7:GFP/nlp7-1 roots (Fig. 2G). These re-sults show that NLP7 is necessary for BLC adhesion.In addition to the border-like cell phenotype, the nlp7-1plant is smaller than the wild type, with a shorterroot and fewer elongated lateral roots (SupplementalFig. S3).We identified another T-DNA insertion mutant line

(SALK_114886) within the last exon of the coding re-gion of NLP7. This allele, nlp7-2, expressed the NLP7transcript in the root at 85% of wild-type levels (15%reduction; Supplemental Fig. S4). Because nlp7-2expressed NLP7 at near wild-type levels and did nothave a defective BLC phenotype, this line was not fur-ther pursued. Thus, the complemented line pNLP7:NLP7:GFP/nlp7-1 was used in addition to nlp7-1 forour experiments.

NLP7 Is Strongly Expressed in BLCs

The above results suggested that NLP7 functions inBLC release and may be important for BLC adhesion.

To further understand the role of NLP7 in BLC release,we examined the expression ofNLP7. Plants expressingpNLP7:GUS showed thatNLP7 is strongly expressed inthe columella root cap and maturation zone of the root(Castaings et al., 2009; Fig. 3A). Examination of thetranslational fusion of pNLP7:NLP7:GFP demonstratedthat NLP7 is expressed in the BLCs under both stan-dard and low pH conditions (Fig. 3, B and C; standardpH shown; low pH in Supplemental Fig. S5). Previouswork showed that NLP7 localizes to both the cytoplasmand nucleus (Castaings et al., 2009; Marchive et al.,2013). We observed NLP7 in the cytoplasm and thenucleus of BLCs, and in the nucleus of cells in the rootdifferentiation zone, in both standard (Fig. 3, B–D) andlow pH conditions (Supplemental Fig. S5).

Mutations in NLP7 Lead to Reduced Pectin, HG, andCellulose Content

Because the nlp7-1 mutant had altered BLC release,and BLC release in Arabidopsis depends on celluloseand pectin content (del Campillo et al., 2004; Durandet al., 2009), we analyzed the levels of cell wall poly-saccharides in the nlp7-1 mutant. We first stained rootsof nlp7-1, the wild type, and the pNLP7:NLP7:GFP/nlp7-1 complemented line with the histochemical stainruthenium red, which stains unesterified (acidic) pectin(Sterling, 1970; Sabba and Lulai, 2002). Roots of thenlp7-1mutant showed less pectin compared to the wildtype at standard pH (Fig. 4, top panel). At low pH,pectin levels in the wild-type root decrease and aresimilar to those in the nlp7-1 mutant (Fig. 4, bottom

Figure 2. Low pH causes single BLC release in Ara-bidopsis. A and B, Wild-type root grown at standard(A) and low pH (B). C and D, nlp7-1 root grown atstandard (C) and low pH (D). E and F, Roots of thecomplemented line (pNLP7:NLP7:GFP in nlp7-1)grown at standard (E) and low pH (F). No GFP is seenin E and F because theGFP filter was not used for theseimages. Blue arrows point to BLCs released as singlecells (B–D) or loose BLC adhesion (F). G, Graphshowing the percentage of roots with BLCs released assingle cells in the wild-type, nlp7-1, and the NLP7complemented line. Different letters indicate signifi-cant differences by ANOVA and Tukey’s HSD test.Bar = 50 mm and is the same for all panels. n. 50 foreach genotype and each condition.

Plant Physiol. Vol. 171, 2016 2103

Genetic Control of Border-Like Cell Release










panel). The pectin content does not further decrease inthe root of nlp7-1 at low pH, possibly because levels inthe mutant are already low (Fig. 4).

HG, a major component of pectin, is the primarypolysaccharide responsible for cell wall adhesion inBLCs of Arabidopsis (Durand et al., 2009). To gainfurther insight into the pectin deficiency in nlp7-1, wenext tested whether HG was altered in the nlp7-1 mu-tant using whole-mount immunolocalization with theHG-specific antibodies JIM5 and JIM7 (Knox et al.,1990; Willats et al., 2000; Clausen et al., 2003; Durandet al., 2009). JIM5 and JIM7 antibodies recognize dif-ferent patterns of methyl esterification on HG (Willatset al., 2000; Clausen et al., 2003). JIM5 binds to bothpartially methyl-esterified HG and unesterified HG,while JIM7 binds only to partially methyl-esterified HGbut does not bind unesterified HG. Consistent with ourruthenium red results, the root cap of the nlp7-1mutantshowed significantly less JIM5 and JIM7 antibody la-beling compared to thewild type at standard pH (Fig. 5;see Supplemental Fig. S6 for negative control with noprimary antibody). To ensure that the differences in JIM5and JIM7 labeling between thewild type and nlp7-1werenot due to differences in antibody penetration betweenthe two genotypes, we used a positive control. Anti-CDPK32 (Calcium-Dependent Protein Kinase 32)showed no difference in labeling between roots of thewild type and nlp7-1 (Supplemental Fig. S6), suggestingthat the differences observed between the wild typeand nlp7-1 for JIM5 and JIM7 are indeed the result ofdecreased pectin in nlp7-1. At low pH, the root tip of

nlp7-1 had decreased JIM7 labeling compared to thewild type, but there was no significant difference inlabeling between thewild type and nlp7-1with the JIM5antibody (Supplemental Fig. S7).

Cellulose, formed by b-1,4-linked glucan chains, isanother major component of plant cell walls. Cellulosedegradation is necessary for proper BLC release (delCampillo et al., 2004). We therefore examined celluloselevels in nlp7-1 seedlings grown at standard pH usingan acid hydrolysis assay (Dubois et al., 1956). Com-pared to the wild type, the nlp7-1 mutant containssignificantly less cellulose (Fig. 6). Together, these ex-periments demonstrate that the nlp7-1 mutant hasdecreased levels of the cell wall polysaccharides pectinand cellulose. This suggests that defective BLC releasein nlp7-1 is due to defects in cell adhesion.

Transcriptional Profiling of nlp7-1 Roots Identifies CellWall-Loosening Enzymes

Given that BLCs were released as single cells innlp7-1, we hypothesized thatNLP7may function in celladhesion in the root cap. We used microarray analysisto investigate whether NLP7 modulated this process.Transcriptional profiling of whole roots of 5 dai nlp7-1plants identified 194 genes differentially expressed(false discovery rate , 0.0001, fold change . 1.5) com-pared to the wild type (Supplemental Table S1). A totalof 160 genes were up-regulated, and 34 were repressedin the root of nlp7-1. Significant Gene Ontology (GO)categories (Provart et al., 2003) for all differentiallyexpressed genes, as well as the up-regulated and down-regulated genes separately, are listed in SupplementalTable S2. Highly significant GO categories for biological

Figure 4. Mutations inNLP7 lead to a decrease in pectin in roots at bothstandard and low pH. Ruthenium red staining of the wild type, nlp7-1,and complemented line (pNLP7:NLP7:GFP/nlp7-1). All roots are 7 dai.Bar = 100 mm; magnification is 203. The experiment was repeatedtwice with n . 10 for each genotype and treatment in each replicate.

Figure 3. NLP7 expression in root. A, Transgenic plants expressingpNLP7:GUS.NLP7 is expressed in the columella root cap as well as theelongation and maturation zone of the root. Bar = 100 mm; 203 mag-nification. B and C, nlp7-1 mutant complemented with pNLP7:NLP7:GFP showing that NLP7 is expressed in cytoplasm (B) and nucleus (C) ofthe BLCs. D, NLP7 expression in the nucleus of the cells in differenti-ation zone of root. White arrow indicates the nucleus and the scale barindicates 50 mm. B to D are 403 magnification.


Karve et al.









process included transport (P = 7.683 1028), response toabiotic/biotic stimulus (P = 1.33 3 1026), response tostress (P = 3.993 1026), and cell organization/biogenesis(P = 1.723 1023). For molecular function GO categories,transporter activity (P = 7.35 3 1024) and DNA/RNAbinding (P = 5.60 3 1024) were most significant. In thecellular component category, the category with thehighest significance (other than unknown)was the Golgiapparatus (P = 5.293 1025). This was interesting becausecell wall polysaccharides such as hemicelluloses aresynthesized in theGolgi and then transported by vesiclesto the cell wall. Genes encoding proteins operating in thecell wall were present but not significant (P = 0.059).Previously, Marchive et al. (2013) identified direct

transcriptional targets of NLP7 in whole nlp7-1 seed-lings that had been resupplied with nitrate for 10 minafter a period of nitrate starvation. We examined

whether any genes differentially expressed in the nlp7-1root were also direct targets of NLP7 in their workand identified 14 genes (Supplemental Table S3). Thisnumber is not higher likely because of the differences ingrowth and environmental conditions in the two datasets: 13-d-old whole nlp7-1 seedlings grown in liquidmedium under N starvation followed by resupply byMarchive et al. (2013) and 5-d-old nlp7-1 roots grown onagar with full nutrient supply in our study. Several ofthe genes that overlapped between the two data setshad roles in nutrient transport (Supplemental Table S3).

Because of our interest in the nlp7-1 border-like cellrelease phenotype, we focused on cell wall-relatedprocesses and identified 11 genes related to the cellwall in our data set. These included CEL5, XTH5, twoexostosins, and a PL-like gene (Supplemental TableS4). All but one of the 11 genes was up-regulated innlp7-1.

We next used the Arabidopsis root expression map(Brady et al., 2007) to examine the cell-type-specific ex-pression pattern of these 11 genes. Due to the defec-tive root cap phenotype, we looked for genes highlyexpressed in the columella and/or lateral root cap. Twogenes, CEL5 (At1g22880) and PL (At1g65570), werehighly expressed in the columella root cap (SupplementalFigure S8), while another, XTH5 (At5g13870), was highlyexpressed in the lateral root cap.

We used qRT-PCR to confirm the expression of thesethree genes in nlp7-1 (Fig. 7). We also examinedexpression of QUASIMODO1 (QUA1) and QUA2.Although these genes were not identified in ourmicroarray analysis, QUA1 and QUA2 encode proteinsthat control pectin biosynthesis and are important forproper border-like cell release (Bouton et al., 2002;Mouille et al., 2007; Durand et al., 2009). As shown inFigure 7A, consistent with our microarray results,CEL5, PL, and XTH5 expression were up-regulatedin the nlp7-1 root, while QUA1 and QUA2 remainedunchanged.

We next used mutant analysis to determine whethermutations in CEL5, XTH5, or PL altered border-like cellrelease. Mutations in each of these genes lead to in-creased root cap layers (Fig. 7B). The cel5 mutant had

Figure 6. Mutations in NLP7 decrease cellulose content in roots. Acidhydrolysis assay showing decreased cellulose content in 5 dai nlp7-1 rootsgrown at standard pH. **P , 0.001 (two-tailed paired t test). Bars in-dicate SD for two biological replicates.

Figure 5. Mutations inNLP7 lead to decreased levels of HG in roots. Aand C, Wild-type; B and D, nlp7-1. Whole-mount immunolabeling ofwild-type and nlp7-1 roots grown at pH 5.7. JIM5 (top panel) and JIM7(middle panel). E and F, Average corrected total fluorescence (CTF) forlabeling intensity of JIM5 (left) and JIM7 (right). nlp7-1 roots show sig-nificantly (P, 0.005) decreased labeling for each. Bar = 50mm. Similarnumbers of slices per z-stack were used for the wild type and nlp7-1 foreach antibody, with each slice 5 mm (see “Materials and Methods”for details). The area extending from the root tip to 200 mmwas selectedfor fluorescence quantification. The experiment was repeated twice,and the average of at least nine seedlings for each genotype and eachantibody is shown. Magnification is 403.











the “stickiest” root cap, and we focused on this gene forfurther analysis. Mutations in CEL5 have been previ-ously shown to lead to defective border-like cell releasein Arabidopsis under standard growth conditions (delCampillo et al., 2004). CEL5 encodes an endo-1,4-b-D-glucanase that hydrolyzes the b-1,4-linked glucanchains that comprise cellulose, and CEL5 expression ishighly enriched in the columella compared to other celltypeswithin the root (Supplemental Fig. S8; del Campilloet al., 2004). As previously observed (del Campillo et al.,2004), analysis of the cel5mutant (T-DNA insertion line

SALK_079921) showed that BLC are not properly re-leased and extra cells stick to the root cap when grownunder standard conditions (del Campillo et al., 2004).We searched for additional cel5 alleles, but found noother lines with significantly reduced CEL5 transcript(Supplemental Fig. S9). Since wild-type plants show anincrease in release of single BLCs when grown underlow pH conditions (Fig. 2), we used confocal micros-copy to examine the root cap of the cel5 mutant afterexposure to low pH. In contrast to the wild type, whenthe cel5 mutant was grown at low pH, the root capretained its stickiness; BLCs adhered to the tip of thecel5 root, and intact root cap layers were clearly ob-served adjacent to the root tip (Fig. 7, C and D). Only14% of cel5 roots showed single border-like cell releaseat low pH compared to 55% of wild-type plants at lowpH. Thus, even in a root cap loosening environment, cellsof the cel5 root cap do not correctly detach.

Single Border-Like Cell Release in nlp7-1 Depends on theExpression Level of CEL5

Because NLP7 encodes a transcription factor, andexpression of CEL5 is activated in the nlp7-1mutant, wehypothesized that CEL5 may be required for the nlp7-1phenotype. To test this, we generated the nlp7-1 cel5double mutant. In roots of the double mutant, BLC re-lease occurs in layers, similar to wild-type plants (Fig.8A), and the average number of root cap layers is notdifferent compared to the wild type (Fig. 8B). Out of13 double mutant lines examined, none showed singleBLC release. Thus, the single BLC release phenotypeobserved in nlp7-1 depends on the expression level ofCEL5.

Expression of Root Cap Regulators Is Altered in nlp7-1

The NAC-domain family transcription factors SMB,BRN1, and BRN2 redundantly regulate root cap matu-ration and cell wall modifications in the root cap(Willemsen et al., 2008; Bennett et al., 2010). The smb-3brn1-1 brn2-1 triple mutant has a very sticky root cap,similar to that of cel5 (del Campillo et al., 2004; Bennettet al., 2010). We used qRT-PCR to assess expressionlevels of these genes in the root of the nlp7-1mutant.Wefound that expression of SMB, BRN1, and BRN2 is ac-tivated (Fig. 9). Expression of CEL5 is reduced in thesmb-3 brn1-1 brn2-1 triple mutant (Bennett et al., 2010).Thus, in mutants with a sticky columella root cap, CEL5expression is reduced, while in the nlp7-1 mutant,which releases BLCs as single cells, CEL5 expression isactivated.

Mutations in NLP7 Lead to Increased Susceptibility to theSoil-Borne Fungus Foc

We hypothesized that the impaired border-like cellrelease in the nlp7-1 mutant may lead to increased

Figure 7. NLP7 regulates border-like cell release by altering expressionof cell wall modifying enzymes. A, Relative expression levels of cellwall-modifying enzymes in nlp7-1mutant by qRT-PCR. B, Total numberof root cap layers in various mutants involved in cell wall modification.C and D, BLCs adhere more tightly to the root cap of the cel5mutant atpH 4.0. C, The 7 dai wild-type root growing at pH 4.0 showing releaseof single border-like cells (white arrow). D, The 7 dai cel5 mutantgrowing at pH 4.0 showing cells adhering to the root cap (white arrow)and several released root cap layers adjacent to the root cap (whitearrowheads). Bar = 50 mm. C and D are 403 magnification. *P , 0.05and **P , 0.001.


Karve et al.





susceptibility to soil-borne pathogens. To test this, weinoculated the soil-borne fungal pathogen Foc directlyon to the tip of nlp7-1 and wild-type roots. Foc firstinvades root systems and then enters the vasculatureand eventually causes wilting of the abovegroundportion of the plant (Tjamos and Beckman, 1989). Asshown in Figure 10, A and B, nlp7-1 has more Focgrowth around the root tips than wild-type or thecomplementation line (Supplemental Fig. S10) due toincreased susceptibility to Foc. Also, fewer nlp7-1roots grew through the Foc inoculation site (Fig.10C). Furthermore, nlp7-1 showed increased antho-cyanin accumulation in the shoots compared to thewild type at 5 d after infection (Fig. 10D). Thus, inkeeping with a role for BLCs in plant defense, muta-tions inNLP7 lead to increased susceptibility to a root-invading pathogen.

DISCUSSION

Collectively, our data support a model in which thetranscription factor NLP7 regulates BLC release bysuppressing expression of cell wall-loosening enzymesas well as key transcription factors involved in root capmaintenance and maturation (Fig. 11). This promotescellulose and pectin maintenance, preventing single-cell BLC release in wild-type plants. Whether NLP7directly regulates these genes or does so indirectlythrough the transcription factors SMB, BRN1, or BRN2is not clear.In wild-type plants, environmental conditions that

lead to cell wall loosening, such as those encountered

under low pH, result in an increase inNLP7 expression.Increased NLP7 expression suppresses cell wall loos-ening by repressing expression of cell wall modificationenzymes, which results in proper cell adhesion understressful conditions. In the nlp7-1mutant, an increase inexpression of cell wall modification enzymes in the rootcap coupled with a decrease in HG and cellulose leadsto release of BLCs as single cells.

Border cells and BLCs are known for their protectiveroles in plant defense (Hawes et al., 2000; Watson et al.,2015). For example, border cells in pea protect the roottip from infection by the pathogenic fungus Nectriahematococca (Gunawardena and Hawes, 2002), whilethose of maize (Zea mays) exude a compound thatpromotes branching in the arbuscular mycorrhizalfungus Gigaspora gigantea (Nagahashi and Douds,2004). In Medicago truncatula, border cells have ele-vated levels of defense compounds (Watson et al.,2015). In Arabidopsis, BLCs perceive and activatedefense signaling in response to microbe-associatedmolecular patterns, such as flagellin22 and peptido-glycan (Plancot et al., 2013). Consistent with a role forBLCs in plant defensemechanisms, mutations inNLP7lead to an increase in susceptibility to the soil-borne,root-infecting fungal pathogen Foc. The increased re-lease of single BLCs from the root cap and the decreasein pectin and cellulose content may facilitate fungalentrance and increase colonization of the mutant root.Given the ubiquity of soil-borne pathogens (Lewis andPapavizas, 1991; Lumsden et al., 1995), this suggeststhat NLP7 may act to protect the root from bioticstress.

The low pH-induced release of BLCs as single cellsmay be a result of changes to the cell wall polysac-charide pectin (Koyama et al., 2001; Fig. 4). Low pHchanges the availability of nutrients in growth mediaand soils, causing a major decrease in the availabilityof Ca++ (Truog, 1946). Arabidopsis roots grown inminimal media at low pH had decreased viabilityafter 2 h, but this effect was ameliorated if the minimal

Figure 9. Quantitative RT-PCR analysis of BRN1, BRN2, and SMB ex-pression reveals that these genes are activated in nlp7-1. Asterisks in-dicate significance: *P, 0.05 and **P, 0.01 (two-tailed paired t test).Error bars show SD.

Figure 8. nlp7-1mutant phenotype depends on expression of CEL5. A,Arrows point to border-like cells or cell layers (in cel5). Border-like cellsof the nlp7-1mutant are released as single cells, while those of the cel5mutant stick to the root cap. The double mutant phenocopies the wildtype. B, Average number of root cap layers in each genotype in A. Bar =50 mm. n . 13 for each genotype; 403 magnification. Letters indicatesignificant differences by ANOVA and Tukey’s HSD test.






media was supplemented with Ca++ (Koyama et al.,2001). Ca++ is a divalent cation with a large ionic ra-dius that can cross-link and stabilize pectic polysac-charides (Carpita and Gibeaut, 1993). Although ourplants are grown on full nutrient agar, since low pHdecreases the availability of Ca++, the low pH-inducedrelease of single BLCs in wild-type plants may be dueto a decrease in available Ca++ and, thus, a decrease inpectin stabilization. This phenotype is exacerbatedat low pH in the nlp7-1 mutant, in which the expres-sion of cell wall-loosening enzymes, including PL, isactivated.

NLP7 is part of the RWP-RK transcription factorfamily, so named for the conserved RWPYRK proteinmotif present in all members (Schauser et al., 2005;Chardin et al., 2014). RWP-RK proteins have been bestdescribed for their roles in nitrogen (N)-regulatedpathways (Ferris and Goodenough, 1997; Schauseret al., 1999, 2005; Borisov et al., 2003; Camargo et al.,2007; Lin and Goodenough, 2007; Marsh et al., 2007). InArabidopsis, NLPs, including NLP7, are master regu-lators of the nitrate response (Castaings et al., 2009;Konishi and Yanagisawa, 2013; Marchive et al., 2013;

Chardin et al., 2014). Members of this family bind toa nitrogen response cis-element to control nitrate-regulated transcription (Konishi and Yanagisawa,2013). The NLP7 protein moves from the cytoplasm tothe nucleus after resupply of nitrate to N-starvedseedlings (Marchive et al., 2013), and nitrate sensingis defective in the nlp7mutant (Castaings et al., 2009).In addition to its role in the N response, nlp7 mu-tants have high drought tolerance (Castaings et al.,2009), suggesting that this transcription factor hasadditional unexplored roles in environmental stressresponses.

Here, we show that NLP7 controls BLC release byregulating cell wall loosening in the root cap. The roleof NLP7 in BLC release appears to be primarilythrough suppression of the enzymes that controlcellulose and pectin degradation. For example, apectin lyase-like gene, which degrades pectin, isup-regulated in the nlp7-1 mutant, but there is nochange in expression of the pectin biosynthetic en-zymes QUA1 and QUA2. Interestingly, cell wallloosening and degradation are important parts of thenodulation process, and recent work has demon-strated that NIN, a NLP7 homolog, binds to thepromoter and activates the expression of a pectatelyase required for nodulation in L. japonicus (Xie et al.,2012). Together, these results suggest that the regu-lation of cell wall modification may be a commonfeature of NIN and NLPs, yet employed in differentdevelopmental contexts. It will be intriguing to de-termine whether additional members of the NLPfamily have roles in BLC release or cell wall modifi-cation in different tissues.

Figure 11. Model for NLP7 control of border-like cell release. See textfor details.

Figure 10. Mutations in NLP7 lead to increased susceptibility to Focinfection. A, Foc growth around the root of root-tip inoculatedwild-typeand nlp7-1 at 48 hpi. B, Quantification of the area of Foc growth aroundthe root tip at 48 hpi. nlp7-1 shows significantly more growth of Focaround the root tip. P, 0.05. C, Percentage of roots growing throughthe inoculation site. D, Graph showing total anthocyanin contentfrom the shoots of the wild type and nlp7-1 at 6 d postinoculation.Letters indicate significant difference using ANOVA and Tukey’sHSD test.


Karve et al.



MATERIALS AND METHODS

Plant Material and Growth Conditions

Seeds of Arabidopsis (Arabidopsis thaliana) wild type (COL-0), nlp7-1(Salk_026134), nlp7-2 (SALK_114886), cel5 (Salk_079921), xth5 (SALK_057512),and a pectin lyase-like gene (GK100C05) were obtained from the ArabidopsisBiological Resource Center. Homozygous nlp7-1 plants were backcrossed onceto Col-0. The seeds were surface sterilized in 50% bleach and stratified at 4°C for48 h. The seedswere plated on standardMurashige and Skoog (MS; pH = 5.7) oron low pHMS (pH= 4.0) plates. The seedlingswere grown in a growth chamberat 22°C under 50% relative humidity with a 16-h/8-h day/night schedule.Unless otherwise noted, plants in all experiments were examined 7 d afterimbibition, and error bars in figures show SD.

To generate complementation lines, 3 kb upstream of the NLP7 start site(NLP7 promoter) was first cloned into vector pDONRP4-P1 using Gatewaytechnology (Life Technologies) to generate pENTR:pNLP7. The coding re-gion of At4g24020 (NLP7) was amplified from cDNA and cloned into Gate-way vector pDONR221 to generate pENTR:NLP7. The vectors pENTR:pNLP7, pENTR:NLP7, and GFP in pDONRP2R-P3 were recombined withpDESTR4-R3 using Multisite Gateway cloning technology (Life Technolo-gies). The final construct of pNLP7:NLP7:GFP was transformed into Arabi-dopsis by the floral dip method (Clough and Bent, 1998). Transgenic plantswere confirmed by PCR using GFP-specific primers, confocal microscopy forGFP expression, and qRT-PCR for NLP7 expression. The seeds of pNLP7:GUS from Castaings et al. (2009) were obtained as a gift fromDr. Anne Krapp(INRA, France).

RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR

For RNA extraction, seedlings were either grown continuously on controlMS plates (pH = 5.7) or transferred to low pH 4.0 plates for 24 h after initialgrowth on control MS as specified for each experiment. Whole seedlings oronly roots were frozen in liquidN2 and stored at280°C until further use at thetime specified for each experiment. Total RNA was isolated using the plantRNA purification kit (Norgen Biotek). On-column DNase treatment wasperformed with DNAse I in DNase digestion buffer (Omega Bio-tek). ThecDNAwas synthesized using 1 mg of total RNAwith oligo(dT) primers usingAMV first-strand cDNA synthesis kit (New England Biolabs) according to themanufacturer’s protocol. The cDNAwas diluted 1:5 (v/v) and 2 mL of dilutedcDNA was used for qRT-PCR. The qRT-PCR was performed using Sso FastEva Green Supermix (Bio-Rad) according to the manufacturer’s instructions.The efficiency of each primer pair set was evaluated using a cDNA dilutionseries, and the relative expression was calculated using the delta-delta Ctmethod. Gene expression was calculated relative to AT1G13320 (Czechowskiet al., 2004) as described by Iyer-Pascuzzi et al. (2011). A total of three to fiveindependent biological replicates, each with three technical replicates, wereperformed to calculate relative fold difference in gene expression. Primersused are listed in Supplemental Table S5. In all qRT-PCR figures, error barsshow SD.

Microarray Sample Preparation and Analysis

Sample preparation and microarray analysis was performed as described(Iyer-Pascuzzi et al., 2011). Briefly, seedlings of the wild type (Col-0) and nlp7-1were grown on MS, pH 5.7, for 5 d and roots cut just below the root-hypocotyljunction. Cut roots were immediately placed into RLT buffer (Qiagen) and RNAextracted using the RNeasy plant mini kit (Qiagen). Two biological replicateswere performed. Probes for array analysis were prepared using the one-cycleamplification protocol from Affymetrix. Samples were hybridized to ATH1microarrays by Expression Analysis. All arrays were normalized and differ-entially expressed probe sets identified using a mixed-model ANOVA Perlscript as described (Levesque et al., 2006). Differentially expressed probe setswere identified using an |1.5| fold change cutoff and a false discovery rate(q-value) of 1 3 1024 as described (Iyer-Pascuzzi et al., 2011). Heat maps werecreated using TMV microarray software (www.tm4.org).

GUS and Ruthenium Red Staining

The seedlings expressing pNLP7:GUS were grown on control MS plates(pH = 5.7) for 6 to 8 d. The seedlings were transferred to either low pH plates

(pH 4.0) or to control plates (pH 5.7). After 24 h, seedlings were incubated inGUS staining buffer containing 50mMNaPO4, pH 7.0, 2.5 mM K3CN6, 2.5 mM

K4CN6, 0.1%, Triton X-100, and 2.5 mM X-gluc for 1.5 h at 30°C and washedwith the GUS staining buffer without X-gluc. To analyze pectin content inthe roots of wild-type and nlp7-1 seedlings, 0.05% (w/v) solution of ru-thenium red (Sigma-Aldrich) was prepared and seedlings were stained for10 min as described by Durand et al. (2009). The pictures were taken with aNikon Eclipse 800 under 203 magnification.

Confocal Microscopy and Immunofluorescence

Seeds of the wild type (Col-0), nlp7-1, cel5, nlp7-1 cel5, xth5, and pl weresterilized as described and seedlingswere grown for 7 d. Seedlings were stainedwith propidium iodide (Sigma-Aldrich) for about 1min andmounted on a glassslide, and roots caps were visualized under a confocal microscope. For all theexperiments, images were taken using Nikon A1Rsi confocal microscope, andimages were edited using NIS elements software (Nikon).

For immunofluorescence experiments, 7 dai wild-type and nlp7-1 seedlingswere fixed in 4%N-N-dimethylformamide for 30 min followed by three washeswith 13 PBS. To prevent nonspecific antibody binding, seedlings were incu-bated in blocking solution (3% nonfat dairymilk in 13 PBS) for 30min followedby three washes with 13 PBS. The seedlings were incubated overnight withprimary antibody JIM5 or JIM7 (CarboSource) diluted 1:6 in 13 PBS with 3%nonfat dairy milk in 13 PBS, washed with TPBS (0.05% Tween in 13 PBS) for20 min with 43 5-min washes, and then incubated with FITC-conjugated sec-ondary antibody diluted 1:50 in TPBS for 1 h at room temperature. Finally, theseedlings were washed four times with TPBS for a total of 20 min followed by alast wash with PBS. Seedlings were left in PBS until confocal microscopy wasdone. A “no primary antibody” control was used in each biological replicate asa negative control to check for specificity of antibodies. As a positive control,both wild-type and nlp7-1 roots were labeled with anti-CDPK32 (custom madeby YenzymAntibodies). The images of roots were taken using aNikon confocalA1Rsi with Z-stack sectioning, and a 3D image was reconstructed using NISelements. Two independent biological replicates were performed. Z-stackprojections for the wild type and nlp7-1 were composed of similar numbers ofslices, and each slice was 5 mm. For fluorescence measurements, the maximumintensity projections were generated using NIS elements ND2 viewer, andfluorescence intensities were measured using ImageJ software. For measuringfluorescence, the area between the root tip and 200 mm upwards was selectedin each root and the outline for the area of the root was drawn in ImageJ. The corrected total fluorescence (CTF) was calculated as CTF = integrateddensity 2 (area of selected root tip 3 mean fluorescence of backgroundreadings). The average of at least nine roots with similar number of Z-stacksections was used for each wild type and nlp7-1 for each antibody. Error barsshow SD.

Quantification of Root Cap Layers

Root cap layerswere counted from the root cap images takenusing a confocalmicroscope. The number of root cap layers was counted from the columellainitials to the last attached layer of BLCs still adhered to the primary root. Aminimum of 30 roots was examined per genotype with at least two biologicalreplicates.

Cellulose Assay

Cellulose content in the nlp7-1mutant and wild type was determined by theacid hydrolysis method modified from Dubois et al. (1956). Briefly, the roots of7 dai nlp7-1 and the wild type were ground in liquid N2 and 3.5 mg each of thefreeze dried powder was used for further analysis. The ground tissue wastreated with trifluoroacetic acid for 90 min at 120°C. To determine the cellulosecontent as a Glc derivative, the phenol-sulfuric acid hydrolysis method wasperformed, and the Glc concentration was determined using a standard curveof 0 to 1,000 nmols of Glc at 500 nm. The entire experiment was repeated twice.Error bars show SD.

Pathogen Assay

nlp7-1 and wild-type plants were grown on standard MS plates for 7 d. Theroot tips of nlp7-1 and the wild type were inoculated with 2 mL of the 1 3 108

microconidia suspension in sterile water of Foc (NRRL #38297). Foc cultureswere grown on potato dextrose agar plates for 3 to 4 d to obtain conidia. The





http://www.tm4.org


plates inoculated with Foc were scanned at 24 and 48 h postinoculation (hpi).Mycelial growth was measured at 48 hpi using Image J. The area of mycelialgrowth around each root of the wild type and nlp7-1was selected in ImageJ andmeasured. Root growth inhibition was measured by counting the number ofroots passing through the infection site. Anthocyanin concentrations weremeasured by standard anthocyanin spectrophotometric assay as described bySims and Gamon (2002). Briefly, shoot tissue of the infected plants was har-vested in liquid N2 and ground in acidified methanol (99% methanol and 1%HCl). The extracts were centrifuged at 4,000g for 5 min at 4°C and the super-natant was used for spectrophotometry. Absorbance for anthocyanin wasmeasured at 530 nm. The total anthocyanin content was calculated by ac-counting for degraded chlorophyll content.

Accession Numbers

Sequence data from this article can be found in the EMBL/GenBank datalibraries under accession number GSE63474.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Time course of gene expression of genes in theSTOP1 cluster after exposure to pH 4.6.

Supplemental Figure S2. qRT-PCR of NLP7 expression in whole roots 24 hafter exposure to pH 4.0 or pH 5.7 shows that NLP7 is activated by lowpH.

Supplemental Figure S3. nlp7-1 mutant phenotype in the whole plant.

Supplemental Figure S4. Levels of NLP7 transcript in nlp7-1 (SALK_026134) and nlp7-2 (SALK_114886) mutants and in the complementedline.

Supplemental Figure S5. NLP7 localization at low pH.

Supplemental Figure S6. Negative and positive controls for immunolabel-ing experiment.

Supplemental Figure S7. Immunolabeling of 5 dai root tips of wild-typeand nlp7-1 seedlings grown at pH 4.0.

Supplemental Figure S8. Heat map of root cell-type-specific expression of11 genes with roles in cell wall-related processes and differentiallyexpressed in nlp7-1 roots compared to the wild type.

Supplemental Figure S9. Semiquantitative RT-PCR for reduction in CEL5transcript in two SALK T-DNA insertion lines.

Supplemental Figure S10. nlp7-1 and the complementation line (pNLP7:NLP7:GFP) 48 hpi with Foc.

Supplemental Table S1. One hundred and ninety four genes differentiallyexpressed between whole roots of nlp7-1 and wild-type Col-0, ATH1microarray, pH 5.7.

Supplemental Table S2. GO categories of the genes differentiallyexpressed in the nlp7-1 root.

Supplemental Table S3. Genes differentially expressed in the nlp7-1 rootand direct targets of NLP7 after nitrate resupply in Marchive et al.(2013).

Supplemental Table S4. Eleven genes with roles in cell wall-related pro-cesses and differentially expressed in the nlp7-1 root.

Supplemental Table S5. Primers used in this study.

Supplemental Methods.

ACKNOWLEDGMENTS

We thank members of the Iyer-Pascuzzi lab for critical reading of themanuscript, members of the Carpita lab for technical help with the celluloseacid hydrolysis assay, Anne Krapp for use of the pNLP7:GUS line, and PhilipBenfey for supporting early experiments with NLP7.

Received March 20, 2016; accepted May 19, 2016; published May 24, 2016.

LITERATURE CITED

Barlow P (2003) The root cap: cell dynamics, cell differentiation and capfunction. J Plant Growth Regul 21: 261–286

Benfey PN, Scheres B (2000) Root development. Curr Biol 10: R813–R815Bennett T, van den Toorn A, Sanchez-Perez GF, Campilho A, Willemsen

V, Snel B, Scheres B (2010) SOMBRERO, BEARSKIN1, and BEARSKIN2regulate root cap maturation in Arabidopsis. Plant Cell 22: 640–654

Bennett T, van den Toorn A, Willemsen V, Scheres B (2014) Precisecontrol of plant stem cell activity through parallel regulatory inputs.Development 141: 4055–4064

Blancaflor EB, Fasano JM, Gilroy S (1998) Mapping the functional roles ofcap cells in the response of Arabidopsis primary roots to gravity. PlantPhysiol 116: 213–222

Blancaflor EB, Fasano JM, Gilroy S (1999) Laser ablation of root cap cells:implications for models of graviperception. Adv Space Res 24: 731–738

Borisov AY, Madsen LH, Tsyganov VE, Umehara Y, Voroshilova VA,Batagov AO, Sandal N, Mortensen A, Schauser L, Ellis N, TikhonovichIA, Stougaard J (2003) The Sym35 gene required for root nodule devel-opment in pea is an ortholog of Nin from Lotus japonicus. Plant Physiol 131:1009–1017

Bouton S, Leboeuf E, Mouille G, Leydecker MT, Talbotec J, Granier F,Lahaye M, Höfte H, Truong HN (2002) QUASIMODO1 encodes a pu-tative membrane-bound glycosyltransferase required for normal pectinsynthesis and cell adhesion in Arabidopsis. Plant Cell 14: 2577–2590

Brady SM, Orlando DA, Lee JY, Wang JY, Koch J, Dinneny JR, Mace D,Ohler U, Benfey PN (2007) A high-resolution root spatiotemporal mapreveals dominant expression patterns. Science 318: 801–806

Camargo A, Llamas A, Schnell RA, Higuera JJ, González-Ballester D,Lefebvre PA, Fernández E, Galván A (2007) Nitrate signaling by theregulatory gene NIT2 in Chlamydomonas. Plant Cell 19: 3491–3503

Carpita NC, Gibeaut DM (1993) Structural models of primary cell walls inflowering plants: consistency of molecular structure with the physicalproperties of the walls during growth. Plant J 3: 1–30

Castaings L, Camargo A, Pocholle D, Gaudon V, Texier Y, Boutet-MerceyS, Taconnat L, Renou JP, Daniel-Vedele F, Fernandez E, Meyer C,Krapp A (2009) The nodule inception-like protein 7 modulates nitratesensing and metabolism in Arabidopsis. Plant J 57: 426–435

Chardin C, Girin T, Roudier F, Meyer C, Krapp A (2014) The plantRWP-RK transcription factors: key regulators of nitrogen responses andof gametophyte development. J Exp Bot 65: 5577–5587

Clausen MH, Willats WG, Knox JP (2003) Synthetic methyl hexagalacturonatehapten inhibitors of anti-homogalacturonan monoclonal antibodies LM7,JIM5 and JIM7. Carbohydr Res 338: 1797–1800

Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743

Czechowski T, Bari RP, Stitt M, Scheible WR, Udvardi MK (2004) Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors:unprecedented sensitivity reveals novel root- and shoot-specific genes.Plant J 38: 366–379

del Campillo E, Abdel-Aziz A, Crawford D, Patterson SE (2004) Root capspecific expression of an endo-beta-1,4-D-glucanase (cellulase): a newmarker to study root development in Arabidopsis. Plant Mol Biol 56:309–323

Driouich A, Follet-Gueye ML, Vicré-Gibouin M, Hawes M (2013) Rootborder cells and secretions as critical elements in plant host defense.Curr Opin Plant Biol 16: 489–495

Dubois M, Gilles K, Hamilton J, Rebers P, Smith F (1956) Colorimetricmethod for determination of sugars and related substances. Anal Chem28: 350–356

Durand C, Vicré-Gibouin M, Follet-Gueye ML, Duponchel L, Moreau M,Lerouge P, Driouich A (2009) The organization pattern of root border-like cells of Arabidopsis is dependent on cell wall homogalacturonan.Plant Physiol 150: 1411–1421

Eapen D, Barroso ML, Ponce G, Campos ME, Cassab GI (2005) Hydro-tropism: root growth responses to water. Trends Plant Sci 10: 44–50

Fendrych M, Van Hautegem T, Van Durme M, Olvera-Carrillo Y, HuysmansM, Karimi M, Lippens S, Guérin CJ, Krebs M, Schumacher K, Nowack MK(2014) Programmed cell death controlled by ANAC033/SOMBRERO deter-mines root cap organ size in Arabidopsis. Curr Biol 24: 931–940

Ferris PJ, Goodenough UW (1997) Mating type in Chlamydomonas isspecified by mid, the minus-dominance gene. Genetics 146: 859–869


Karve et al.



















Gunawardena U, Hawes MC (2002) Tissue specific localization of rootinfection by fungal pathogens: role of root border cells. Mol Plant Mi-crobe Interact 15: 1128–1136

Hawes MC, Brigham LA, Wen F, Woo HH, Zhu Y (1998) Function of rootborder cells in plant health: pioneers in the rhizosphere. Annu RevPhytopathol 36: 311–327

Hawes MC, Gunawardena U, Miyasaka S, Zhao X (2000) The role of rootborder cells in plant defense. Trends Plant Sci 5: 128–133

Hawes MC, Lin HJ (1990) Correlation of pectolytic enzyme activity withthe programmed release of cells from root caps of pea (Pisum sativum).Plant Physiol 94: 1855–1859

Iuchi S, Koyama H, Iuchi A, Kobayashi Y, Kitabayashi S, Kobayashi Y,Ikka T, Hirayama T, Shinozaki K, Kobayashi M (2007) Zinc finger proteinSTOP1 is critical for proton tolerance in Arabidopsis and coregulates akey gene in aluminum tolerance. Proc Natl Acad Sci USA 104: 9900–9905

Iyer-Pascuzzi AS, Jackson T, Cui H, Petricka JJ, Busch W, Tsukagoshi H,Benfey PN (2011) Cell identity regulators link development and stressresponses in the Arabidopsis root. Dev Cell 21: 770–782

Knox JP, Linstead PJ, King J, Cooper C, Roberts K (1990) Pectin esterifi-cation is spatially regulated both within cell walls and between devel-oping tissues of root apices. Planta 181: 512–521

Kochian LV, Piñeros MA, Liu J, Magalhaes JV (2015) Plant adaptation toAcid soils: the molecular basis for crop aluminum resistance. Annu RevPlant Biol 66: 571–598

Konishi M, Yanagisawa S (2013) Arabidopsis NIN-like transcription fac-tors have a central role in nitrate signalling. Nat Commun 4: 1617

Koyama H, Toda T, Hara T (2001) Brief exposure to low-pH stress causesirreversible damage to the growing root in Arabidopsis thaliana: pec-tin-Ca interaction may play an important role in proton rhizotoxicity. JExp Bot 52: 361–368

Kumpf RP, Nowack MK (2015) The root cap: a short story of life and death.J Exp Bot 66: 5651–5662

Levesque MP, Vernoux T, Busch W, Cui H, Wang JY, Blilou I, Hassan H,Nakajima K, Matsumoto N, Lohmann JU, Scheres B, Benfey PN (2006)Whole-genome analysis of the SHORT-ROOT developmental pathwayin Arabidopsis. PLoS Biol 4: e143

Lewis JA, Papavizas GC (1991) Biocontrol of plant disease: the approachfor tomorrow. Crop Prot 10: 95

Lumsden RD, Lewis JA, Fravel DR (1995) Formulation and delivery ofbiocontrol agents for use against soilborne plant pathogens. In F Hall, JBarry, eds, Biorational Pest Control Agents, Formation and Delivery.American Chemical Society, Washington DC, pp 166-182

Li X, Ma J, Hiradate S, Matsumoto H (2000) Mucilage strongly bindsaluminum but does not prevent roots from aluminum injury in Zea mays.Physiol Plant 108: 152–160

Lin H, Goodenough UW (2007) Gametogenesis in the Chlamydomonasreinhardtii minus mating type is controlled by two genes, MID andMTD1. Genetics 176: 913–925

Marchive C, Roudier F, Castaings L, Bréhaut V, Blondet E, Colot V,Meyer C, Krapp A (2013) Nuclear retention of the transcription factorNLP7 orchestrates the early response to nitrate in plants. Nat Commun4: 1713

Marsh JF, Rakocevic A, Mitra RM, Brocard L, Sun J, Eschstruth A, LongSR, Schultze M, Ratet P, Oldroyd GE (2007) Medicago truncatula NINis essential for rhizobial-independent nodule organogenesis induced by

autoactive calcium/calmodulin-dependent protein kinase. Plant Physiol144: 324–335

Mouille G, Ralet MC, Cavelier C, Eland C, Effroy D, Hématy K,McCartney L, Truong HN, Gaudon V, Thibault JF, Marchant A, HöfteH (2007) Homogalacturonan synthesis in Arabidopsis thaliana requires aGolgi-localized protein with a putative methyltransferase domain. PlantJ 50: 605–614

Nagahashi G, Douds DD Jr (2004) Isolated root caps, border cells, andmucilage from host roots stimulate hyphal branching of the arbuscularmycorrhizal fungus, Gigaspora gigantea. Mycol Res 108: 1079–1088

Plancot B, Santaella C, Jaber R, Kiefer-Meyer MC, Follet-Gueye ML,Leprince J, Gattin I, Souc C, Driouich A, Vicré-Gibouin M (2013)Deciphering the responses of root border-like cells of Arabidopsis andflax to pathogen-derived elicitors. Plant Physiol 163: 1584–1597

Provart NJ, Gil P, Chen W, Han B, Chang HS, Wang X, Zhu T (2003) Geneexpression phenotypes of Arabidopsis associated with sensitivity to lowtemperatures. Plant Physiol 132: 893–906

Sabba RP, Lulai EC (2002) Histological analysis of the maturation of nativeand wound periderm in potato (Solanum tuberosum L.) Tuber. Ann Bot(Lond) 90: 1–10

Schauser L, Roussis A, Stiller J, Stougaard J (1999) A plant regulatorcontrolling development of symbiotic root nodules. Nature 402: 191–195

Schauser L, Wieloch W, Stougaard J (2005) Evolution of NIN-like proteinsin Arabidopsis, rice, and Lotus japonicus. J Mol Evol 60: 229–237

Sims DA, Gamon JA (2002) Relationships between leaf pigment contentand spectral reflectance across a wide range of species, leaf structuresand developmental stages. Remote Sens Environ 81: 337–354

Sterling C (1970) Crystal-structure of ruthenium red and stereochemistry ofits pectic stain. Am J Bot 57: 172–175

Tjamos EC, Beckman CH (1989) Vascular Wilt Diseases of Plants. Springer-Verlag, Berlin

Truog E (1946) Soil reaction influence on availability of plant nutrients. SoilScience Society Proceedings 11: 305–308

Vicré M, Santaella C, Blanchet S, Gateau A, Driouich A (2005) Rootborder-like cells of Arabidopsis. Microscopical characterization and rolein the interaction with rhizobacteria. Plant Physiol 138: 998–1008

Watson BS, Bedair MF, Urbanczyk-Wochniak E, Huhman DV, Yang DS,Allen SN, Li W, Tang Y, Sumner LW (2015) Integrated metabolomicsand transcriptomics reveal enhanced specialized metabolism in Medi-cago truncatula root border cells. Plant Physiol 167: 1699–1716

Wen F, Zhu Y, Hawes MC (1999) Effect of pectin methylesterase gene ex-pression on pea root development. Plant Cell 11: 1129–1140

Willats WG, Limberg G, Buchholt HC, van Alebeek GJ, Benen J, ChristensenTM, Visser J, Voragen A, Mikkelsen JD, Knox JP (2000) Analysis of pecticepitopes recognised by hybridoma and phage display monoclonal antibodiesusing defined oligosaccharides, polysaccharides, and enzymatic degradation.Carbohydr Res 327: 309–320

Willemsen V, Bauch M, Bennett T, Campilho A, Wolkenfelt H, Xu J,Haseloff J, Scheres B (2008) The NAC domain transcription factors FEZand SOMBRERO control the orientation of cell division plane in Ara-bidopsis root stem cells. Dev Cell 15: 913–922

Wolf S, Mouille G, Pelloux J (2009) Homogalacturonan methyl-esterificationand plant development. Mol Plant 2: 851–860

Xie F, Murray JD, Kim J, Heckmann AB, Edwards A, Oldroyd GE,Downie JA (2012) Legume pectate lyase required for root infection byrhizobia. Proc Natl Acad Sci USA 109: 633–638





Documents

The Transcription Factor NIN-LIKE PROTEIN7 Controls Border-Like