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Received 29 Apr 2013 | Accepted 16 Oct 2013 | Published 15 Nov 2013

DOI: 10.1038/ncomms3779 OPEN

Arabidopsis SABRE and CLASP interact to stabilize cell division plane orientation and planar polarity

Stefano Pietra1, Anna Gustavsson1, Christian Kiefer1, Lothar Kalmbach1,w, Per Hrstedt2, Yoshihisa Ikeda1,w, Anna N. Stepanova3, Jose M. Alonso3 & Markus Grebe1

The orientation of cell division and the coordination of cell polarity within the plane of the tissue layer (planar polarity) contribute to shape diverse multicellular organisms. The root of Arabidopsis thaliana displays regularly oriented cell divisions, cell elongation and planar polarity providing a plant model system to study these processes. Here we report that the SABRE protein, which shares similarity with proteins of unknown function throughout eukaryotes, has important roles in orienting cell division and planar polarity. SABRE localizes at the plasma membrane, endomembranes, mitotic spindle and cell plate. SABRE stabilizes the orientation of CLASP-labelled preprophase band microtubules predicting the cell division plane, and of cortical microtubules driving cell elongation. During planar polarity establishment, sabre is epistatic to clasp at directing polar membrane domains of Rho-of-plant GTPases. Our ndings mechanistically link SABRE to CLASP-dependent microtubule organization, shedding new light on the function of SABRE-related proteins in eukaryotes.

1 Ume Plant Science Centre, Department of Plant Physiology, Ume University, SE-90 187 Ume, Sweden. 2 Ume Core Facility Electron Microscopy, Ume University, SE-90 187 Ume, Sweden. 3 Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695, USA. w Present addresses:

Department of Plant Molecular Biology, University of Lausanne, UNIL-Sorge, Biophore Building, 1015 Lausanne, Switzerland (L.K.); Centre of the Region Han for Biotechnological and Agricultural Research, Department of Molecular Biology, Faculty of Science, Palack University Olomouc,lechtitel 11, 78371 Olomouc, Czech Republic (Y.I.). Correspondence and requests for materials should be addressed to M.G. (email: mailto:[email protected]

Web End [email protected] ).

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Diverse multicellular organisms coordinate the polarity of single cells within the plane of the tissue layer15. This type of tissue polarity is commonly referred to as planar

polarity1,2,4. The establishment of planar polarity is intensively studied for animal epithelia where Drosophila wing-hair polarization provides an invaluable model system1,2. Similarly, plants like Arabidopsis display planar polarity of coordinated polar hair outgrowth from root epidermal cells3,4 and orientation of leaf epidermal hairs (trichomes)5. The upstream molecular mechanisms that direct planar polarity, however, differ substantially between animals and plants. In Drosophila, Wingless and its homologue dWnt4 act as long-range polarizing cues upstream of the Frizzled planar cell polarity pathway6, which transmits directional information to downstream effectors such as small Rho/Rac-type family GTPases1,2. By contrast, plants lack genes with similarity to Wnts or to core components of the Frizzled planar cell polarity pathway. Instead, planar polarity of root epidermal cells is coordinated by a tissue concentration gradient of the plant hormone auxin4,7. This gradient instructs the coordinated polar placement of hairs and of Rho-of-plant (ROP) GTPases that mark the initiation of planar polarity of root hair positioning before morphological hair outgrowth4,7. ROP2/4 localization provides the earliest read-out for polar hair initiation8,9 and regulation of ROP activity spatially restricts polar root hair initiation9,10. During morphogenesis of leaf pavement cells, auxin can activate ROP2/4 and ROP6 to differentially organize the actin and tubulin cytoskeleton, respectively11,12. Actin and tubulin are also required for planar polarity of root hair initiation13,14, but cytoskeletal dynamics and their regulation remain uncharacterized during this process. Similarly, the sole microtubule-associated protein known to be required for planar orientation of leaf trichomes is the CLIP170-associated protein (CLASP) from Arabidopsis15. Further, CLASP mediates orientation of cell division planes in roots16,17, modulates the abundance18,19 as well as the polarity of PIN2 protein in root cortical cells19, and CLASP homologues regulate cellular asymmetries in other eukaryotes20. At the molecular level, CLASP homologues associate with and stabilize the plus ends of dynamic microtubules20, stabilize microtubule arrays during cell division15,16,20 and promote microtubule growth around cell edges contributing to the generation of plant cortical microtubule arrays21.

Here we report new functions for the Arabidopsis SABRE (SAB) gene, mechanistically linking its action to CLASP-mediated microtubule organization. We thus unravel new interactions contributing to the framework of cell division plane orientation and planar polarity formation in plants. Our ndings may help to elucidate the function of uncharacterized SAB-like proteins found throughout eukaryotes and their potential function in cytoskeletal organization during cell division, cell morphogenesis and its coordination.

Resultskreuz und quer affects cell and planar polarity. The root hairs of Arabidopsis regularly emerge from the outer membrane of epidermal cells and coordinately initiate close to, albeit not completely at, the root tip-oriented (basal) ends of cells (Fig. 1a). As knowledge about genes contributing to planar polarity in plants remains limited, we performed a genetic screen for root hair-positioning mutants to identify additional factors. This led to the identication of the recessive kreuz und quer (kuq) mutant generated by fast neutron mutagenesis. kuq displayed a more random distribution of hair position towards both the basal and the apical ends of cells (Fig. 1b,c). We also observed the localization of polar ROP protein patches at the outer membrane of root hair cells

before morphological hair outgrowth. Unlike wild type (Fig. 1d), kuq mutant cells often showed more basal or apical localization of ROP (Fig. 1eg). In contrast, the polar distribution of PEN3-GFP (Supplementary Fig. S1a,b) that broadly denes the outer membrane22,23 as well as the apical polarity of the PIN2 auxin efux carrier (Supplementary Fig. S1c,d) remained largely unaffected in root epidermal cells of kuq (Supplementary Fig. S1b,d). More strikingly, the polarity of GFP-BASL24, which almost invariably localizes to basal membranes and to nuclei when ectopically expressed in wild-type root epidermal cells(99.8% basal, 0.2% of cells apical localization; n 514) was

signicantly more often switched to apical ends of epidermal cells in kuq (8.6% of cells with apical localization; n 187;

P 5.1 10 9 by Fishers exact test; Supplementary Fig. S1e,

f). These ndings suggest that kuq affects basal membrane polarity and basal bias of planar polarity rather than outer lateral membrane polarity in the root epidermis.

Leaf trichome orientation provides another model for the analysis of both cell and planar polarity. Arabidopsis wild-type trichomes predominantly carry three branches, and the rst branch, initiating closest to the leaf surface, is oriented towards the proximal end of the leaf5,15 providing a planar polarity readout. Intriguingly, orientation of the rst branch proved much more variable in the kuq mutant than in the wild type (Fig. 1hj), revealing defective planar polarity of trichome orientation. At the cellular level, kuq mutant leaves showed two-branched trichomes more often than wild-type leaves (Fig. 1k,l; for quantitative analysis see below) and sometimes displayed irregular placement of branches along the trichome stalk (Fig. 1m). The reduction in branch number and the ectopic placement of branches on trichomes in kuq indicated a cell polarity defect in the initiation and placement of trichome branches. Interestingly, reduced trichome branch number has been reported for Arabidopsis mutants defective in tubulin and microtubule-associated proteins25. Hence, kuq affects cell and planar polarity in leaf and root epidermis, causing phenotypes characteristic of microtubule cytoskeleton mutants.

kuq is allelic to SABRE. We mapped the kuq mutation to a 480-kb interval on chromosome 1 by positional cloning (Fig. 2) and analysed phenotypes of T-DNA insertion lines in candidate genes within this interval. Homozygous seedlings from 9 out of 11 T-DNA insertion lines in one of these genes, SABRE (SAB) (At1g58250; Fig. 2) displayed phenotypes indistinguishable from that of kuq, whereas two showed similar but weaker seedling root and adult shoot defects (Figs 2 and 3a). Sequencing of the genomic SAB coding region in kuq identied a single-nucleotide deletion at position 6,592 (of 7,824), predicted to induce a nonsense mutation generating a premature stop codon (Fig. 2). Other SAB-mutant alleles have been described to display anisotropic expansion of root cortical cells2628 or an enhanced response to phosphate starvation29. More detailed analysis of three SAB T-DNA insertion lines, SALK 108709 (sab-5), SALK 52995 (sab-6) and SALK 123771 (sab-7) uncovered reduced epidermal cell length and defective planar polarity of root-hair position similar to that of the kuq mutant (Fig. 3be). Complementation analyses revealed that these three lines were allelic to kuq and to each other and they are, therefore, in the following referred to as sabkuq, sab-5, sab-6 and sab-7. The sab-7 mutant carrying an insertion into the second last exon of the SAB gene (Fig. 2) was somewhat less affected, identifying it as a hypomorphic allele (Fig. 3a,b,e). Reverse transcription PCR (RTPCR) analyses revealed that neither sab-5, sab-6 nor sab-8 (SALK 12897) produced a full-length transcript, in contrast to sabkuq for which a transcript could be detected when using primers spanning the

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Figure 1 | kuq mutation affects epidermal cell and planar polarity. (a,b) Hair-forming root epidermal cells of 5-day-old (a) wild-type Columbia (Col) and (b) kuq seedlings. Arrowheads, apical and basal ends of cells in a, b and df. Asterisks indicate sites of hair formation in a and b. (c) Quantitative analysis of planar polarity phenotypes as relative root hair position in Col and kuq. Number of cells in classes (frequency) with hair positions are indicated between basal (0) and apical (1) ends of cells (**P 0.000 Col versus kuq, determined by non-parametric, two-sample KolmogorovSmirnov (KS) test,

n 150 cells from 30 roots per genotype). (df) anti-ROP (red) immunouorescence in Col, (e,f) kuq epidermal cells, combined with DAPI-stained nuclei

(blue) and brighteld microscopy image. (g) Quantitative analysis of planar polarity phenotypes of ROP protein patch positions along apical-basal axis of epidermal cells in Col and kuq. Number of cells in classes (frequency) showing ROP positions between basal (0) and apical (1) ends is shown (**P 0.000

Col versus kuq by KS test, n 50 cells per genotype, from 10 Col and 19 kuq roots). (h,i,km) Scanning electron microscopy (SEM) images of leaf

trichomes of (h,k) Col, (i,l,m) kuq-mutant leaves. (h,i) Planar polarity of rst trichome branch orientation along proximo-distal leaf axis (arrows). Distal ends of leaves are to the left, proximal ends to the right. Deviation of o30 and 430 from proximal orientation is indicated by green and red arrows, respectively. (j) Quantitative analysis of rst trichome branch orientation from SEM images of Col and kuq. 0 indicates proximal orientation, 180 distal orientation of trichome branches. (**Po0.001 Col versus kuq by F-test, n 174 trichomes per genotype). (km) Trichome branching phenotypes of (k) Col

and (l,m) kuq. Note, (l) trichomes with two branches or (m) additional ectopically placed branch in kuq (arrowhead). For statistical analysis, see Fig. 5a. Scale bars, 50 mm (a,b), 10 mm (df), 200 mm (h,i), 100 mm (km).

region including the single-nucleotide deletion (Fig. 3fh). We subsequently employed sab-5 in parallel to or instead of sabkuq because sab-5 carried the T-DNA insertion closest to the 50-end of the SAB coding region (Fig. 2), did not express full-length SAB complementary DNA (cDNA) (Fig. 3fh) and displayed phenotypic defects similar to sabkuq (Fig. 3ac, Supplementary Fig. S2al). Thus, cloning of sabkuq revealed a central role for SAB in cell and planar polarity.

SABRE-like proteins in other eukaryotes. Our sequencing of SAB wild-type cDNA amplied from Arabidopsis roots experimentally veried the presence of an 8,347-bp transcript encoding a 2,607 amino acid protein identical to the predicted protein sequence At1g58250.1 (ref. 29). Depending on the transmembrane helix prediction algorithm employed, SAB is predicted to contain one (TMHMM, Pred-TMR), two (TMpred) or three transmembrane domains (TopPred2, HMMTOP). All programs predict the rst six to eight amino-terminal amino acids preceding the rst transmembrane domain to be cytosolic. This indicates that SAB contains an internal, hydrophobic start-transfer sequence rather than an N-terminal cleavable signal peptide and that SAB may be a transmembrane protein rather than a secreted protein. Proteins with sequences related to the predicted 2,607 amino acid SAB protein are found throughout diverse eukaryotes (Fig. 4a). In Arabidopsis thaliana, KINKY

POLLEN (KIP) shares the highest sequence identity with SABRE28. Although plant species harbour either one or two SAB-like genes in their genomes, all animal genomes analysed, including the human and mouse genomes, possess a single-copy gene encoding a protein of similar size with highest sequence identity restricted to two larger protein regions (Fig. 4b). To date, none of the genes related to SAB from non-plant eukaryotes has been functionally characterized. However, mutants defective in the Arabidopsis KIP gene and the closely related ABERRANT POLLEN TRANSMISSION1 (APT1) gene from maize are defective in pollen tube growth28,30. APT1, like SAB, codes for a predicted 2,607 amino acid protein30. When transiently expressed in tobacco pollen, a carboxy-terminal 108 amino acid peptide from APT1 fused to green uorescent protein (GFP) localizes to the Golgi apparatus30. The peptide has been annotated as a Golgi-body localization domain in the conserved domain database31. However, although SAB is a predicted membrane protein with the N terminus oriented towards the cytosol, the biological and molecular function of SAB and related proteins as well as the subcellular localization of full-length proteins remain to be addressed in any eukaryote.

SABCLASP interaction in planar polarity. As the SAB sequence did not provide information about the molecular function of the protein, we addressed the mechanism of SAB

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SAIL 425 G04

FLAG 378H03

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... AGA ATC TCC TAT TTA GAG GTG TAC TTT TTC GAG TTC

. R L P Y I E V H F L E L

AAT CTC CTA TTT AGA GGT GTA CTT TTT GAG TTC

STOP L I F R W M F F E L

Figure 2 | kuq causes a single-nucleotide deletion in the SABRE gene. Map-based identication of the kuq mutation in the SAB gene. Simple-sequence-length polymorphism (SSLP; NGA111), custom-made SSLPs (Monsanto Insertion/Deletion polymorphism; MID) and custom-made cleaved amplied polymorphic sequence (CAPS; MSNP) markers are indicated with respective position, recombinant/total chromosomes and recombination frequency. Digits of MID and MSNP markers correspond to polymorphism accession number in Monsanto Arabidopsis Polymorphism Collection (http:// www.arabidopsis.org/browse/Cereon/index.jsp). Note the 480-kb mapping interval containing JAtY63C14 transformation-competent bacterial articial chromosome carrying the SAB gene. Insertion positions of 11 SAB T-DNA lines analysed are indicated by arrowheads below the gene structure. Black boxes, exons; lines, introns; orange boxes, untranslated regions. T-DNA lines in black, homozygous seedlings phenotypically indistinguishable from kuq mutants; T-DNA lines in grey, weaker phenotype. T-DNA lines with green arrowheads, tested for allelism to kuq by complementation test. Red circle, cytosine deletion in kuq inducing a premature stop codon in 17th exon of SAB; upper rows, wild-type coding sequence and amino-acid translation; lower rows, kuq.

action by analysing its genetic interactions. The combination of trichome branching and orientation defects observed in sab mutants had been described for clasp mutants15. We therefore generated a sab-5;clasp-1 double mutant and observed a strongly enhanced trichome branching defect, when compared with the respective single mutants or sabkuq (Fig. 5a; Supplementary Fig. S3a,b), suggesting that SAB and CLASP synergistically interact during placement of trichome branches. Similarly, root length and overall growth of sab-5;clasp-1 double-mutant seedlings was severely reduced compared with the single mutants, further pointing towards a synergistic interaction at this level (Fig. 5b; Supplementary Fig. S3c).

We next addressed whether CLASP acts on planar polarity of root hair placement and observed a clear basal shift of hair position in clasp-mutant alleles (Fig. 5ce). Similarly, ROP protein patches were misplaced towards the basal-most ends of elongating clasp-mutant epidermal cells when compared with the

wild type (Fig. 5fh), demonstrating that CLASP acts on planar polarity before ROP positioning. Strikingly, ROP positioning in sab-5;clasp-1 double-mutant seedlings was indistinguishable from that of sab-5 (Fig. 5ik), suggesting that sab is epistatic to clasp during polar ROP positioning.

SABCLASP interaction during division plane orientation. The genetic interactions between SAB and CLASP prompted us to investigate whether SAB contributes to other processes that involve CLASP function, such as orientation of the cell division plane. Strikingly, sab-5- and sabkuq-mutant root meristems displayed strongly aberrantly orientated cell division planes when compared with the wild type (Fig. 6a,b; Supplementary Fig. S3d,e). Live imaging of microtubules and nuclear DNA co-visualized by mCherry-TUBULIN ALPHA-5 (mCherry-TUA5)32,33 and HISTONE2B-YELLOW FLUORESCENT

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Figure 3 | Phenotypic and RTPCR analyses of additional SAB T-DNA-mutant alleles. (a) Five-week-old plants of Col, sab-7, sabkuq and sab-5. Note, sab-5 is indistinguishable from sabkuq while sab-7 is less affected. (b) Quantitative analysis of trichoblast length in Col and SAB-mutant alleles. Average cell length of n 150 trichoblasts from 30 roots per genotype. Data are meanss.d. from n 3 independent experiments. Statistical differences determined by two-

tailed, unpaired t-test with equal variance and signicance level Po0.05 are indicated as *Po0.05 and **Po0.01; n 3. All sab alleles differ signicantly

from wild type but do not differ from each other (P40.05). (ce) Quantitative analysis of planar polarity defects in (c) sab-5, (d) sab-6 and (e) sab-7 compared with Col is displayed as relative root hair position similar to Fig. 1c. **P 0.000 Col versus sab-5, sab-6 or sab-7 by KS test, n 150 cells from 30

roots per genotype. (fh) Semiquantitative RTPCR analysis of wild-type Col and sab-mutant seedlings. SAB transcript was amplied with (f) primers spanning the T-DNA insertion (or single-nucleotide deletion) and primers located close to (g) the 50- or (h) the 30-end of SAB coding sequence. Primers close to 30-end were also employed on genomic DNA (gDNA) extracted from the same plant material used for cDNA (cDNA) synthesis. Partial SAB transcripts were detected in all T-DNA alleles. EF1-a and APT1 were utilized as controls. Scale bar, 10 mm.

PROTEIN (H2B-YFP)34, respectively, revealed that in comparison with wild type (Fig. 6c), sab-5-mutant cells displayed misorientation of the preprophase band (PPB; Fig. 6d). This orientation defect was followed by similarly misaligned spindle and phragmoplast orientation throughout mitosis and cytokinesis, whereas the overall timing of mitosis and cytokinesis was indistinguishable from wild-type root cells (Fig. 6d; Supplementary Movies 1 and 2). Similar results were obtained in immunolocalization studies demonstrating that PPB, spindle and phragmoplast orientation were defective in epidermal cells of both sab-5- and sabkuq-mutant root meristems (Fig. 6eq; Supplementary Fig. S3fh). PPB, spindle and phragmoplast orientation in the sab-5;clasp-1 double mutant did not statistically differ from the sab-5 single mutant, but were signicantly more strongly affected than in the clasp-1 single mutant (Fig. 6oq). Thus, SAB function is required for cell division plane orientation already before or at the level of PPB orientation and sab shows epistasis over clasp during this process. Further, in comparison with wild type (Fig. 6r), the orientation of functional GFP-CLASP21 on sab-5 mutant PPBs was affected and coincided with the misoriented alignment of PPB microtubules

(Fig. 6s,t). These results suggested that SAB function is required for orientation of CLASP-guided cortical microtubules and stabilizes correct placement of PPB microtubules in the future plane of cell division.

SAB acts on cortical microtubule arrays. We next analysed potential effects of SAB on cortical microtubule orientation in late meristematic epidermal cells at the transition to cell elongation, in rapidly expanding cells, and during later stages of hair cell differentiation. Strikingly, in contrast to the transversal orientation of mCherry-TUA5-marked microtubules observed in both late meristematic cells (Supplementary Fig. S4a) and in rapidly elongating wild-type cells (Fig. 7a), sab-5-mutant cells of the same size displayed a more random orientation of cortical microtubules than wild-type cells in both cases (Supplementary Fig. S4b,c; Fig. 7b,c). We further monitored the directionality of microtubule growth using the microtubule plus end-tracking protein ( TIP)

AtEB1a-GFP35 in combination with mCherry-TUA5. This revealed bidirectional movement of transversally oriented cortical microtubules in elongating wild-type cells and of the

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Query

1 500 1,000 1,500 2,000 2,500 kuq

Arabidopsis thaliana SABRE (1) Arabidopsis thaliana KIP (2)

Zea mays APT1 (6)

Oryza sativa (5)

Selaginella moellendorffii (7) Physcomitrella patens (8)

Apis mellifera (17)

Oryza sativa (3)

Zea mays (4)

Ostreococcus tauri (9)

Drosophila melanogaster (19)

Rattus norvegicus (10)

Pongo abelii (12)

Mus musculus (11)

Xenopus tropicalis (15)

Caenorhabditis elegans (20)

Homo sapiens KIAA0100 (13)

Gallus gallus (14)

Ciona intestinalis (16)

Anopheles gambiae (18)

Saccharomyces cerevisiae (21) Saccharomyces cerevisiae (22) Schizosaccharomyces pombe (23)

Saccharomyces cerevisiae (21) NP_015442.1 Saccharomyces cerevisiae (22) NP_013559.1 Schizosaccharomyces pombe (23) NP_594183.2

Ostreococcus tauri (9) XP_003080077.1

Oryza sativa (5) ABF95966.1Zea mays APT1 (6) NP_001106066.1 Oryza sativa (3) ABF98216.1

Arabidopsis thaliana SABRE (1) NP_176121.3 Arabidopsis thaliana KIP (2) NP_001078738.1

Selaginella moellendorffii (7) XP_002969704.1

Physcomitrella patens (8) XP_001779593.1

Zea mays (4) DAA50499.1

Caenorhabditis elegans (20) NP_493625.4 Apis mellifera (17) XP_624880.3 Drosophila melanogaster (19) NP_647789.2 Anopheles gambiae (18) XP_315477.2 Ciona intestinalis (16) XP_002122817.1 Danio rerio (24) NR_023325.1Xenopus tropicalis (15) XP_002937671.1 Gallus gallus (14) XP_415819.3

Homo sapiens KIAA0100 (13) NP_055495.2

Rattus norvegicus (10) XP_220636.3 Mus musculus (11) NP_001002004.2

0.1

Pongo abelii (12) XP_002827215.2

Figure 4 | SAB-related proteins in diverse eukaryotes. (a) Blastp search for proteins homologous to SAB and alignment of SAB-like protein sequences from selected organisms. Note, sequence similarity with proteins from more distantly related species is restricted to two distinct regions of about 600 and 300 amino acids. Red arrow, position of premature stop codon introduced by kuq mutation. Numbers in parentheses refer to sequences in b. Red, alignments with highest sequence identity; black, lowest sequence identity. (b) Phylogenetic tree based on sequences of SAB-related proteins in selected species, showing evolutionary conservation of SAB across a wide variety of eukaryotic organisms. NCBI reference sequence or GenBank accession number for each protein are indicated next to the respective species.

more randomly oriented microtubules in sab-5-mutant epidermal cells (Fig. 7a,b; Supplementary Movies 3 and 4). Intriguingly, elongating trichoblasts just before hair initiation displayed a reoriented, bipolar arrangement of longitudinally growing microtubules (Fig. 7d; Supplementary Movie 5), where the majority of microtubule plus ends grew with apical directionality at apical ends of cells (Fig. 7e) and with basal directionality at basal ends of cells (Fig. 7f). This bipolarity of microtubule growth resembled the one recently described for elongating hypocotyl cells36. The overall longitudinal orientation and bipolarity of microtubule growth was less clearly axially aligned in sab-5-mutant cells (Fig. 7g; Supplementary Movie 6). In addition, mCherry-TUA5 co-visualized with the root hair initiation site marker PIP5K3-YFP37 revealed an accumulation

of radially reorganized cortical microtubules at the root hair initiation site in 45% of the cells observed (32/71; Fig. 7hl; Supplementary Fig. S4d,e), whereas 55% did not harbour such an array (Supplementary Fig. S4f,g). The majority of cells (94%, 67/71) at this differentiation stage showed longitudinally oriented microtubules interspersed in diffuse, cytosolic mCherry-TUA5 uorescence indicative of microtubule depolymerization and reorganization in these cells (Fig. 7i; Supplementary Fig. S4dg). In comparison, only a minority of cells (6%, 4/71) still revealed transversal microtubule orientation, when hair initiation sites were already marked by PIP5K3-YFP. Although we did occasionally observe reorganized cortical microtubules at PIP5K3-YFP-marked hair initiation sites in sab-5-mutant cells (Fig. 7mo), none of these cells (0%, 0/50) displayed one unique

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** ** **

120

sab-5; clasp-1

100

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Percentage of trichomes

60%

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100%

** clasp-1 clasp-2

80%

sab-5

40%

20%

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Col

clasp-1

sabkuq

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sab-5;

clasp-1

Col *

Clasp-1

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4 branches 3 branches 2 branches

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sab-5; clasp-1

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Figure 5 | SABCLASP interaction mediates planar polarity upstream of ROP. (ak) Genetic action of sab and interaction with clasp during (a) leaf trichome branching, (b) seedling development and (ck) planar root epidermal polarity establishment. (a) Percentage of 2-branched (red), 3-branched (green) and 4-branched (blue) trichomes in Col (total trichome number: n 545), clasp-1 (n 537), kuq-mutant allele of SAB (sabkuq; n 409), sab-5

(n 498) and sab-5;clasp-1 (n 431) obtained from 1016 leaves per genotype. Note, signicant differences between all genotypes except for sab-5 and

sabkuq (P 0.51); the percentage of 2-branched trichomes in sab-5;clasp-1 highly signicantly differed from the sum of the respective single-mutant values

(dotted line, arrowhead; Po0.001). Signicances were determined by Fishers exact test with signicance level Po0.05 and are indicated as **Po0.01. Supplementary Table S1 shows exact P-values. (b) Ten-day-old Col, clasp-1, sab-5 and sab-5;clasp-1 seedlings. (c,d) Root hair position on epidermal cells of 5-day-old (c) Col and (d) clasp-1 seedlings. (e) Quantitative analysis of planar polarity in Col, clasp-1 and clasp-2 is displayed as relative root hair position as in Fig. 1c. (**P 0.000 Col versus clasp-1; **P 0.000 Col versus clasp-2 by KS test; n 150 cells per genotype). (f,g,i,j) Planar polarity phenotypes

revealed by anti-ROP immunolabelling (red), DAPI (blue) overlaid with brighteld image of epidermal cells from (f) Col, (g) clasp-1, (i) sab-5 and (j) sab-5;clasp-1. (h,k) Quantitative analysis of polar ROP protein patch positions as in Fig. 1g. (h) Col, clasp-1, clasp-2, (k) clasp-1, sab-5 and sab-5;clasp-1(**P 0.004 Col versus clasp-1; **P 0.000 Col versus clasp-2; P 0.241 clasp-1 versus clasp-2; **P 0.000 clasp-1 versus sab-5; **P 0.000 clasp-1

versus sab-5;clasp-1; P 0.841 sab-5 versus sab-5;clasp-1 by KS test, n 50 cells from 8 to 22 roots per genotype). Arrowheads, apical and basal ends of

cells in c, d, f, g, i and j. Asterisks indicate sites of hair formation in c and d. Scale bars, 10 mm (b), 50 mm (c,d), 10 mm (f,g,i,j).

radial rearrangement that could be unambiguously identied as preceding a hair initiation site comparable to the situation in wild type (Fig. 7mo; Supplementary Fig. S4hk). Hence, because of the irregular cortical microtubule arrangement in sab-5, microtubule organization did not allow for prediction of the positions of hair initiation sites. However, PIP5K3-YFP-marked hair initiation sites occurred in ectopic basal-most, or more apical positions along sab-5-mutant hair cells (Fig. 7mp; Supplementary Fig. S4hk), when compared with the wild type (Fig. 7hj,p; Supplementary Fig. S4dg). These ndings suggest that SAB activity is required for cortical microtubule reorganization at correct sites before hair outgrowth. Thus, SAB function mediates transversal microtubule alignment in elongating cells and polar placement of domains of reorganizing microtubules in cells initiating hair formation.

SAB subcellular localization. We next determined the sub-cellular localization of the predicted full-length 290 kDa SAB protein encoded by a messenger RNA of low abundance27,28. To this end, we employed recombineering38 to insert three

consecutive copies of the cDNA encoding for the bright uorescent protein Ypet (3xYpet) at the 50- or at the 30-end of the genomic coding sequence of SAB. Both constructs rescued the overall sab-5 growth defect and all phenotypes investigated, including cell division plane orientation and planar polarity of root hair initiation (Fig. 8ag). The planar polarity phenotype of sab-5 was fully rescued by SAB-3xYpet and largely, albeit not completely, by 3xYpet-SAB (Fig. 8f,g). Both constructs were expressed at low levels throughout the root tip and displayed indistinguishable subcellular distribution (Figs 8hl and 9a,b). These results suggested a likely orientation of both the N- and the C-terminus towards the cytosol because uorescence of cell wall-oriented GFP and YFP protein tags has been reported to be quenched by the low cell wall pH (refs 39,40). 3xYpet-SAB and SAB-3xYpet localized in patches at the cell periphery, suggesting localization to the plasma membrane or cell wall and to endomembrane compartments (Figs 8k,l and Fig. 9a,b). Plasmolysis experiments in the presence of a cell wall marker revealed retraction of SAB-3xYpet from the cell wall (Fig. 9c,d). Strikingly, meristematic cells showed clear enrichment of

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Figure 6 | sab is epistatic to clasp at mediating plane orientation. (a,b) Cell division orientation in root meristematic cells of 5-day-old (a) Col and (b) sab-5 seedlings. Cells are outlined by FM4-64 uorescence. (c,d) Live imaging of microtubules (mCherry-TUA5, magenta) and DNA (H2B-YFP, green) during cell division in meristems of 5-day-old seedlings. (c) Wild-type (wt; SAB/SAB or SAB/sab-5), (d) sab-5 homozygote (sab-5) cells. Number of cell division experiments analysed: wt, n 8; sab-5, n 7. Time points from preprophase band-(PPB)-marked prophase (00) until the end of cytokinesis (690)

are indicated. Dotted lines, mitotic microtubule array or cell division orientation. (em) Mitotic microtubule array orientation in root meristematic cells are revealed by anti-a-tubulin immunolocalization (magenta). DAPI-labelled DNA, blue. (eg) PPB in (e) wild type Col and (f,g) sab-5. (hj) Metaphase plate in (h) Col and (i,j) sab-5. (km) Late phragmoplast in (k) Col and (l,m) sab-5. Angles of microtubule array orientation were obtained from n 60 cells per

mitotic stage, per genotype Col, clasp-1, sab-5 and sab-5;clasp-1. Images for clasp-1 and sab-5;clasp-1 are not shown. (em) Arrowheads, plane of mitotic microtubule array orientation. (n) Microtubule array deviation from midplane are indicated as 0% (left) and 100% (right). (oq) Quantitative, statistical analyses of anti-a-tubulin immunolocalization. (o) PPB, (p) metaphase plate, (q) late phragmoplast orientation in Col, clasp-1, sab-5 and sab-5;clasp-1. (oq)

Box-and-whiskers plots for n 60 cells per mitotic stage, per genotype. Whiskers, maximum and minimum values of population. Blue boxes, 25% of values

above median. Red boxes, 25% of values below median. Statistical differences between populations determined by KS test are indicated as *Po0.05 and **Po0.01 (signicance level Po0.05). For exact P-values, see Supplementary Table S2. Note, sab-5;clasp-1 does not signicantly differ from sab-5 in oq, but signicantly differs from Col and clasp-1 in o and q. (rt) Orientation (arrowheads) and co-localization (white) of GFP-CLASP (green) with mCherry-

TUA5 (magenta) at PPBs of (r) wild-type (wt; SAB/SAB or SAB/sab-5) and (s,t) sab-5 homozygotes (sab-5). (s,t) Note, aberrant PPB orientation in sab-5. Scale bars, 50 mm (a,b), 5 mm (cm,rt).

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Figure 7 | SAB is required for cortical microtubule orientation during cell elongation and planar polarity. (a,b) Live imaging of cortical microtubule arrays in early elongation stage root hair cells co-expressing mCherry-TUA5 (magenta) and the plus end-tracking protein AtEB1a-GFP (green). Note, (a) transversal orientation in wild type (wt; SAB/SAB or SAB/sab-5), versus (b) more random orientation in sab-5 homozygote (sab-5). (c) Quantitative analysis of microtubule angles relative to the apical-basal cell axis in hair cells of 2636 mm length from phenotypically wt and sab-5 seedlings. Statistical analysis reveals highly signicant difference between wt and sab-5 (**P 0.000 by KS test, n 673 microtubules analysed for wt and n 802 for

sab-5, from 28 and 20 cells). For similar results on late meristematic cells of 1318 mm length, see Supplementary Fig. S4ac. (dg) Live imaging of cortical microtubule arrays in later elongation stage root hair cells co-expressing mCherry-TUA5 (magenta) and the plus end-tracking protein AtEB1a-GFP (green).

(d) Bipolar axial orientation of microtubule growth in wt cell just before rst hair formation. (e,f) Magnications of regions marked in d, showing plus ends of microtubules preferentially growing towards (e) apical and (f) basal ends of cell. (g) Perturbed axial alignment and growth of microtubulesin sab-5-mutant cell. (ho) Live co-imaging of mCherry-TUA5-labelled microtubules with hair initiation site marker PIP5K3-YFP. (h) PIP5K3-YFP,(i) mCherry-TUA5, (j) merge of PIP5K3-YFP (green) and mCherry-TUA5 (magenta) in wt cells. (k,l) Magnications of inset in j, showing (k) PIP5K3-YFP and (l) mCherry-TUA5 at hair initiation site. (m) PIP5K3-YFP, (n) mCherry-TUA5, (o) merge of PIP5K3-YFP (green) and mCherry-TUA5 (magenta) in sab-5. Arrowheads, microtubule (re-)organization at (i) close-to-basal site in wt versus (n) basal-most or apically shifted sites in sab-5. (p) Quantitative analysis of polar PIP5K3-YFP patch position, marking site of root hair initiation. Number of cells in classes (frequency) showing PIP5K3-YFP position between basal (0) and apical (1) ends of epidermal cells is shown (**P 0.000 wt versus sab-5 by KS test, n 50 cells per genotype from 39 wt and 41

sab-5 roots). For additional microtubule visualization, see Supplementary Fig. S4dk. Scale bars, 5 mm.

SAB-3xYpet at domains of apical and basal plasma membranes that had just started to retract from the cell wall at the onset of plasmolysis (Fig. 9c). Similarly, transversal, apical-basal membranes of non-plasmolysed cells showed an enrichment of SAB-3xYpet (Fig. 9a) and 3xYpet-SAB co-localization with the plasma membrane marker NSPN12-mCherry/WAVE131R (ref. 41; Fig. 9e). Co-visualization of SAB-3xYpet with endoplasmic reticulum (ER; Fig. 9f), Golgi (Fig. 9g) and trans-Golgi network (TGN) (Fig. 9h) markers41,42 revealed that the endomembrane compartments containing 3xYpet-SAB/SAB-3xYpet did neither represent ER, Golgi nor TGN (Fig. 9fh). Our ndings do not support the previously posed hypotheses that full-length SAB-like proteins may be Golgi-localized or secreted at steady state, respectively28,30. Rather, our results provide in vivo evidence corroborating the identication of SAB peptides from plasma membrane preparations by proteomic approaches43. Further, SAB-3xYpet accumulated in small patches at the nuclear envelope where it co-labelled with microtubules marked by

mCherry-TUA5 during mitotic prophase (Fig. 9i). At the cell cortex, SAB-3xYpet did not precisely co-localize with PPB microtubules but consistently closely associated with them in the majority of prophase cells analysed (91%, 84/92) (Fig. 9i, arrowheads; Supplementary Fig. S5af). Strikingly, SAB-3xYpet was also found in patches along mitotic spindle microtubules (Fig. 9j,k), located to cell plate membranes from early to late stages of cell plate formation (Fig. 9ln), and subsequently remained at domains of apical and basal membranes of dividing cells (Fig. 9e,n). These ndings were conrmed in co-labelling studies with rhodamine phalloidin for F-actin staining in aldehyde-xed cells (Fig. 9ot) and by co-labelling of the KNOLLE cell plate syntaxin with SAB-3xYpet at the cell plate (Fig. 9u). In elongating epidermal cells, SAB-3xYpet distributed more evenly in endomembrane compartments of unknown identity throughout the cytosol (Fig. 9b). In more differentiated cells, where SAB-3xYpet expression was very low and just detectable above background, SAB-3xYpet compartments were

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Figure 8 | SAB-3xYpet and 3xYpet-SAB rescue sab-5 phenotypes and display identical localization. (a) Five-day-old seedlings, (b) 21-day-old plants of Col, sab-5, SAB-3xYpet;sab-5 and 3xYpet-SAB;sab-5. Note, rescue of sab-5 growth defects. (ce) Cell division orientation in root meristem of 5-day-old (c) Col, (d) SAB-3xYpet;sab-5 and (e) 3xYpet-SAB;sab-5 seedlings. Cells outlined by FM4-64 uorescence. (f,g) Quantitative analysis of planar polarity in (f) SAB-3xYpet;sab-5 and (g) 3xYpet-SAB;sab-5 compared with Col and sab-5 is displayed as relative root hair position similar to Fig. 1c. **P 0.000 Col

versus sab-5; P 0.422 Col versus SAB-3xYpet;sab-5; **P 0.000 sab-5 versus SAB-3xYpet;sab-5; *P 0.019 Col versus 3xYpet-SAB;sab-5; **P 0.000

sab-5 versus 3xYpet-SAB;sab-5 by KS test, n 150 cells from 30 roots per genotype. Note, (f) full rescue of sab-5 planar polarity defects by SAB-3xYpet

and (g) partial rescue by 3xYpet-SAB. (h,i) Expression of SAB-3xYpet throughout the root meristem was detected by immunolabelling with anti-GFP antibody (magenta), DAPI (blue), of (h) Col and (i) SAB-3xYpet;sab-5 seedlings. Images are transversal sections showing SAB-3xYpet signal in all cell layers. (jl) Live imaging of (j) Col, (k) SAB-3xYpet;sab-5 and (l) 3xYpet-SAB;sab-5 seedlings showing indistinguishable subcellular localization of SAB-3xYpet and 3xYpet-SAB in root meristematic epidermal cells. Scale bars, 2 mm (a), 10 mm (b), 50 mm (ce,h,i), 10 mm (jl).

present, although not much enriched, at sites of root hair initiation before morphological outgrowth (Fig. 9v,w). Taken together, we show that SAB localizes to plasma membrane domains, preferentially but not exclusively to apical and basal plasma membranes of meristematic cells, at the nuclear periphery, anking the PPB at the onset of mitosis, to mitotic spindles, to cell plate membranes as well as to as yet unidentied endomembrane compartments. Thus, SAB is present in most root tip cells including dividing, elongating and differentiating epidermal cells, consistent with its functions in epidermal cells reported here.

DiscussionOur study unravels new roles for the large SAB protein related to uncharacterized proteins found throughout eukaryotes. We report important functions for SAB in PPB, spindle, phragmoplast and cell division plane orientation, cortical microtubule orientation and planar polarity establishment. Moreover, we uncover biological functions of SAB in cell and planar polarity that it shares with CLASP. A role for CLASP function during microtubule-dependent PIN2 polarity establishment in cortical cells and PIN2 abundance modulated by endocytosis has recently attracted attention18,19. Although SAB and CLASP do not

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a b c

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Figure 9 | SAB subcellular localization. (ah,v,w) Live imaging of functional SAB-3xYpet or 3xYpet-SAB in root epidermal cells of 5-day-old seedlings. (a,b) SAB-3xYpet in sab-5 background detected in (a) meristematic cells and (b) elongating cells. (c,d) Onset of plasmolysis in 5-day-old, SAB-3xYpet;sab-5 seedling root co-labelled for SAB-3xYpet (green) and cell wall stain propidium iodide (magenta). Note, (c) apical and basal membrane domain labelling (arrowheads) and (d) separation of SAB-3xYpet from cell wall (arrowheads). (eh) Meristematic root epidermal cells. (e) 3xYpet-SAB (green) co-localizes with plasma membrane marker NPSN12-mCherry/WAVE131R (magenta). (fh) SAB-3xYpet (green) co-detected with (f) ER marker ER-ECFP-HDEL (magenta), (g) Golgi marker SYP32-mCherry/WAVE22R (magenta) and (h) TGN marker VHA-a1-mRFP (magenta). Merged channels to the right in eh. Note, (f) little co-localization and (g,h) no co-localization when compared with e. (it) SAB-3xYpet (green) (in) co-expressed with mCherry-TUA5 (magenta) and (ot) co-labelled with rhodamine phalloidin (magenta) for F-actin visualization (RP/F-actin) in paraformaldehyde-xed roots. (i) Prophase. SAB-3xYpet and mCherry-TUA5 uorescence around the nuclear envelope. Note, association of SAB-3xYpet (arrowheads) with PPB but no direct co-localization (see also Supplementary Fig. S5). (o) Late prophase. SAB-3xYpet and RP/F-actin. (j,k) SAB-3xYpet localization at mitotic spindles preferentially towards the periphery (arrowheads) in (j,p) metaphase and (k,q) anaphase. (l,r) Late telophase. (l) SAB-3xYpet in centre of phragmoplast at early cell plate and at remaining spindle microtubules (arrowheads). (r) SAB-3xYpet co-labelling with F-actin at early cell plate. (m,s) Late cytokinesis. SAB-3xYpet at cell plate co-labelling with (m) peripheral phragmoplast microtubules and (s) peripheral phragmoplast F-actin. (n,t) End of cytokinesis. SAB-3xYpet at transversal (apical, basal) plasma membranes. (u) anti-KNOLLE cell plate marker immunouorescence (magenta) and SAB-3xYpet (green) uorescence. Channel merge (lower panel) indicates co-localization at cell plate. (v,w) Elongated epidermal cells undergoing hair initiation and bulge outgrowth, visualized by colour-coded spectral lambda scan between 416 and 686 nm. Ypet-specic uorescence, green. (v) Wild type Col. (w) Genomic SAB-3xYpet;sab-5. Images in aw acquired with LSM780 32-channel gallium arsenide phosphide detector array. Scale bars, 5 mm (a,b,dx), 1 mm (c).

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appreciably affect apical epidermal PIN2 polarity, we uncover that CLASP mediates establishment of planar polarity of ROP and root hair positioning, and we conrm a previously reported role of CLASP in PPB orientation15,16. Intriguingly, however, our results unravel that sab mutations perturb all investigated processes more strongly than clasp mutations. sab affects PPB, spindle and phragmoplast orientation as well as planar root hair polarity more strongly than clasp, and shows an epistatic interaction with clasp. Synergistic action of SAB and CLASP in controlling root length and trichome branching may reect additional nonlinear interaction, including potential feedback regulation. Alternatively, synergistic action can be interpreted by interaction of SAB and CLASP with additional partners during these two processes. However, during PPB, spindle and phragmoplast orientation as well as planar polarity in root epidermal cells, SAB and CLASP act sequentially. Moreover, SAB likely acts upstream of CLASP considering the misorientation of CLASP-labelled PPBs observed in sab, the stronger phenotypes of sab mutants and the epistatic relationship of sab over clasp. Hence, our observations mechanistically link SAB function to CLASP-guided cortical microtubule organization during these processes. We further describe a newly observed bipolar arrangement of longitudinally growing microtubules at later stages of root hair cell elongation. Although striking and similar to the bipolar arrangement recently observed in hypocotyl cells36, the functional importance of this dynamic bipolar reorganization, and how it may affect polar site selection during root hair initiation, remains to be determined. Further, we nd that sab is epistatic to clasp during polar placement of ROP protein patches at sites of hair initiation where we observe cortical microtubule reorganization before morphological onset of hair outgrowth. Earlier observations have implicated microtubules in tip growth of different cell types in various organisms14,4446, and radial microtubule alignment has been observed, for example, in tips of moss protonemata44 as well as in conifer pollen tubes45. In addition, microtubule dynamics are known to have a function in directing the growth of and limiting the number of growth sites in root hairs46,47. Our work also points out a role for microtubule reorganization in polarity of root hair cells. However, we report that microtubules are involved earlier than previously described, namely, during selection of the site of root hair initiation. This suggests that the microtubule cytoskeleton acts both in determining planar polarity of hair cell differentiation and in maintaining polar tip growth of root hairs. It remains to be determined how SAB affects microtubule reorganization at the molecular level because the genetic interactions observed between SAB and CLASP may not necessarily reect direct interaction at the protein level. Yet, SAB-3xYpet localized to the perinuclear region from where microtubules radiate towards the PPB as well as adjacent to the PPB, suggesting that SAB may be, directly or indirectly, involved in nucleation or guidance of PPB microtubules. This localization does, however, not match exactly the distribution of CLASP, which directly co-localizes with PPB microtubules at cell edges and binds to microtubule plus ends20,21. This suggests that the observed genetic interactions may reect an indirect or transient interaction within the same pathway. The SAB protein localizes to spindle microtubules from early prophase, and later on is observed close to the spindle periphery consistent with our ndings that SAB is required for spindle and phragmoplast orientation. Further, the accumulation of SAB at cell plate membranes in the centre of the phragmoplast and on apical and basal plasma membranes opens the door for future studies on the role of SAB at these sites. Hence, our work paves the way to elucidating the molecular function of SAB in microtubule reorganization during cell division plane orientation, cell and planar polarity formation in plants. It also encourages

further studies on the connection between SAB-related proteins and the microtubule cytoskeleton. Future studies may now address what proteins the large membrane protein SAB interacts with at the molecular level and which of the homology regions observed in SAB-like proteins throughout eukaryotes may be required for such interactions. Interestingly, the human genome encodes a single SAB-related predicted 2,235 amino-acid-long protein of unknown function named KIAA0100, which is overexpressed in human cancers and also known as, for example, breast cancer overexpressed1 or monocytic leukaemia-associated antigen22 (refs 4851). Hence, our ndings linking SAB to CLASP-mediated microtubule organization may provide a stepping stone for functional analysis of SAB-related proteins during eukaryotic development and for dissecting their potential role in human cancer.

Methods

Plant growth conditions. Medium and growth conditions were as described4. In brief, seeds were surface sterilized and stratied at 4 C for 3 days before plating on MS plates (1 MS medium, 1% sucrose, 0.8% plant agar, 1 M

morpholinoethanesulphonic acid, pH 5.7). Seedlings were grown vertically at 23 C day and 18 C night under 16 h light/8 h dark photoperiod and subjected to analysis after 5 days.

Plant material and genotyping. Arabidopsis thaliana ecotype Columbia (Col-0) was employed as a wild type and as background for the kuq mutant; the following T-DNA insertion lines from the SALK collection52 were obtained from the Nottingham Arabidopsis Stock Centre: sab-5 (stock number N608709), sab-6 (N552995), sab-7 (N623771) and sab-8 (N512897). All above sab alleles, with the exception of sab-7, were unable to self-fertilize and produced 510 seeds per silique on pollination with wild-type pollen. Lines were maintained as heterozygotes and molecular genotyping was used to identify plants carrying mutant alleles. Other previously described mutants and transgenic lines were provided by the Nottingham Arabidopsis Stock Centre or corresponding authors of the cited publications: pPEN3:PEN3-GFP53, p35S:GFP-BASL24, clasp-1 (refs 15,16) (N620061), clasp-2 (refs 15,16) (N583034), p35S:mCherry-TUA5 (refs 32,33), p35S:H2B-YFP34, pCLASP:GFP-CLASP21, p35S:EB1a-GFP35, pGL2:NTF54, pPIP5K3:PIP5K3-YFP37, pUBQ10:NSPN12-mCherry/WAVE131R41, pUBQ10:SYP32-mCherry/WAVE22R41 and VHA-a1-mRFP42. Other mutant and transgene combinations were generated in this study. Primers for PCR genotyping of mutants are listed in Supplementary Table S3.

Immunouorescence and CLSM. Whole-mount immunolabelling of roots was performed as described4. Antibody dilutions were the following: rabbit anti-ROP 1:25 (ref. 8); rabbit anti-PIN2 1:400 (ref. 55); mouse anti-a-tubulin DM1A 1:200 (Santa Cruz, Sc-32293); rabbit anti-GFP 1:1,000 (Torrey Pines, TP-401); rabbit anti-KNOLLE 1:4,000 (ref. 56); DY649 donkey anti-rabbit 1:800 (Jackson ImmunoResearch) for anti-ROP, anti-PIN2, anti-GFP; DyLight633 donkey anti-rabbit 1:100 (Agrisera, AS122034) for anti-KNOLLE; TRITC donkey anti-mouse 1:150 (Jackson ImmunoResearch). Donkey secondary antibodies for multilabelling pre-absorbed to crossreacting species were employed. DNA was stained with 40,6-diamidino-2-phenylindole (DAPI) (1 mg ml 1, Sigma). Imaging of immunolabelled or live roots was carried out using either a Leica TCS SP2 AOBS (Leica) spectral confocal laser scanning microscopy (CLSM) system mounted on an Leica DM IRE2 inverted microscope or a Zeiss LSM780 spectral CLSM system equipped with a 32-channel gallium arsenide phosphide detector array and mounted on a Zeiss Axio observer Z1 inverted microscope. Laser excitation lines, emission lters and acquisition modes for all uorophore combinations are listed in Supplementary Table S4. Oil-corrected Plan-Apochromat 63 objectives,

numerical aperture 1.4 (HCX PL APO 63.0 1.40 OI BD UV, Leica; or DIC M27,

Zeiss) were used for immunolabelling studies. For live imaging, a water-corrected C-Apochromat 40 objective, numerical aperture 1.2 (M27, Zeiss) was used. It

was not possible to detect 3xYpet-SAB and SAB-3xYpet uorescent fusions with a Leica SP2 confocal microscope in live imaging studies because of extremely low intensity of the signal. Hence, all imaging of these lines was carried out with a Zeiss LSM780 microscope. It was however possible to detect SAB-3xYpet and 3xYpet-SAB with a Leica SP2 microscope after immunolabelling with anti-GFP antibody (Torrey Pines). Images were processed and analysed using ZEN2010 (Zeiss), ImageJ (http://www.imagej.nih.gov/ij/

Web End =http://www.imagej.nih.gov/ij/) or Adobe Photoshop CS4 (Adobe Systems), and assembled in Adobe Illustrator CS3 (Adobe Systems).

Analysis of root hair position and trichoblast length. Quantitative microscopic analysis of root hair position and cell length was performed in three independent experiments as described4. Statistical analysis employed non-parametric KolmogorovSmirnov (KS) test and two-tailed t-test.

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Quantitative analysis of ROP positioning. Relative ROP position in trichoblasts before hair initiation was determined by immunolocalization as described4. Measurements were performed on n 50 cells per genotype, from 8 to 22 roots.

Images were analysed using Leica Application Suite Advanced Fluorescence software (Leica).

Scanning electron microscopy of leaf trichome orientation. Growth of sab mutants was signicantly slower compared with wild-type plants. To analyse leaves at a similar developmental stage, the third true leaf from 21-day-old sab plants was compared with the third true leaf from 14-day-old wild-type plants that did not obviously differ in trichome orientation from 21-day-old wild-type plants. Leaves were xed in 2.5% glutaraldehyde in sodium cacodylate buffer overnight at room temperature. To keep the leaves submerged in the solution, the xation was carried out in a desiccator with a low under-pressure to take away trapped air. Fixed material was dehydrated in increasing ethanol concentrations, up to pure ethanol, and was thereafter dried from liquid CO2 using standard critical point drying technique (Polaron E3000 CPD apparatus, Quorum Technologies Ltd, Ashford, UK). Dried specimens were attached to specimen holders using adhesive carbon tape and thereafter coated with B15 nm gold in an evaporation system. Microscopy was performed in a scanning electron microscope running at standard conditions (Carl Zeiss SMT/Cambridge Instruments 360iXP, Cambridge, UK) and micrographs were recorded at standardized magnications and locations on the leaves. Trichome orientation was investigated in a rectangle of 2 mm 1.5 mm in

the centre of the leaf adaxial surface. Angles between the proximal-distal leaf axis and the trichomes rst branch were measured on scanning electron microscope images using ImageJ.

Isolation and positional cloning of kuq. The kuq mutant was isolated in a genetic screen of 5-day-old seedlings for aberrant planar polarity of root hair positioning. A population of B20,000 M2 seedlings obtained from B2,500 fast-neutron-mutagenized M1 plants (Col-0 ecotype, Lehle Seeds) were grown vertically and screened by stereomicroscopy. kuq was characterized by a more random root hair position along epidermal cells, shorter and radially swollen roots and bent cotyledons. The semi-sterile kuq mutant was fertilized with Col-0 pollen and produced ve to eight seeds, which were subjected to further analyses. The kuq phenotype segregated at 27% (n 496) after backcrossing, indicating that it was caused by

a single, recessive mutation. kuq was crossed to Landsberg erecta (Ler) to generate a mapping population, and genomic DNA (gDNA) was extracted from 1,298 F2 seedlings displaying the kuq phenotype and used for mapping by positional cloning57. SSLP and CAPS molecular markers were generated as described57and by using the Monsanto Arabidopsis Polymorphism Collection (http://www.arabidopsis.org/browse/Cereon/index.jsp

Web End =http:// http://www.arabidopsis.org/browse/Cereon/index.jsp

Web End =www.arabidopsis.org/browse/Cereon/index.jsp ), and the mutation was mapped to a 480-kb interval on chromosome 1 (Fig. 2). Analysis of T-DNA insertion lines in genes within this interval suggested the SABRE (SAB) gene as the strongest candidate, with 9 T-DNA lines whose homozygous seedlings looked indistinguishable from kuq mutants. The entire B14.5 kb SAB genomic region was

PCR amplied from kuq mutants and sequenced with three times coverage. This revealed a single cytosine deletion in the 17th exon of the SAB gene, at position 6,592 (of 7,824) of the coding sequence. The mutation caused a frame shift that was calculated to introduce a stop codon at position 6,6176,619, truncating the predicted SAB protein after amino acid 2,205 (of 2,607). A CAPS marker designed over the mutated site physically conrmed the nucleotide deletion and allowed molecular genotyping of kuq mutants (see Supplementary Table S3).

RTPCR analysis. Total RNA was extracted from roots of 5-day-old seedlings employing the RNeasy Plant Mini Kit (Qiagen) according to manufacturers instructions. Two biological replicates were analysed for each mutant and the respective wild-type control. RNA was puried with the Ambion DNA-free Kit (Ambion), precipitated with LiCl and resuspended in diethyl pyrocarbonate-treated H2O. cDNA was synthesized using iScript cDNA Sythesis Kit (Bio-Rad) and used for semiquantitative RTPCR analysis with primer pairs spanning the T-DNA insertion (or single-nucleotide deletion), and primer pairs located respectively at the 50- and 30-end of the SAB gene (Supplementary Table S3 for primer sequences).

Primer pairs were designed to span introns to identify potential gDNA contaminations. Primers amplifying the ELONGATION FACTOR 1-a (EF1-a) or

ADENINE PHOSPHORIBOSYL TRANSFERASE 1 (APT1) genes were used as controls. Reactions were carried out with 34 cycles for T-DNA insertion spanning primers (22 cycles for EF1-a primers) and 35 cycles for primers located close to the 50- or 30- end of the SAB gene (35 and 40 cycles for APT1 primers, respectively).

Analysis of SAB evolutionary conservation. Searches for proteins with similarity to SAB were performed with Blastp version 2.2.28 (http://www.blast.ncbi.nlm.nih.gov/blast/Blast.cgi

Web End =http://www.blast.nc

http://www.blast.ncbi.nlm.nih.gov/blast/Blast.cgi

Web End =bi.nlm.nih.gov/blast/Blast.cgi ) with default parameters using full SAB sequence (At1g58250.1) against non-redundant protein sequences database. Alignment of Saccharomyces cerevisiae and Schizosaccharomyces pombe proteins was achieved after second PSI-BLAST iteration. Full sequences of SAB-related proteins from selected species were aligned with MUSCLE version 3.8.31 (http://www.ebi.ac.uk/Tools/services/web/toolform.ebi?tool=muscle

Web End =http://www.ebi.ac.uk/ http://www.ebi.ac.uk/Tools/services/web/toolform.ebi?tool=muscle

Web End =Tools/services/web/toolform.ebi?tool=muscle ). Output phenogramme generated

from second iteration was visualized with Phylodendron Phylogenetic tree printer (http://www.iubio.bio.indiana.edu/treeapp/treeprint-form.html

Web End =http://www.iubio.bio.indiana.edu/treeapp/treeprint-form.html).

Trichome branching analysis. Measurements of trichome branching were performed as described15. Third true leaf of 21-day-old plants was observed with a Leica MZ 9.5 stereomicroscope (Leica), and number of branches was countedfor all trichomes on adaxial leaf surface. Number of leaves (plants) analysed are as follows: 10 for Col-0, 12 for clasp-1, 16 for sabkuq, 16 for sab-5 and 16 for sab-5;clasp-1.

FM4-64 staining. Seedlings were incubated in 1 MS medium with 25 mM FM4-

64 (Molecular Probes) for 5 min on ice. After two washes with 1 MS medium on

ice, seedlings were mounted on cover slips in 1 MS medium.

Live imaging of microtubules and nuclei during cell division. Wild-type looking and sab-5 seedlings expressing mCherry-TUA5 (ref. 32) and H2B-YFP34 were imaged live. A region of 53.14 mm 53.14 mm centred on a meristematic cell in

prophase (displaying clear PPB) was visualized at intervals of 1 min throughout cell division. For each time point, Z stacks of six optical sections at 0.46 mm distance were acquired, and the image closest to the centre of the cell was selected. Imaging windows and planes were constantly adjusted to account for movement of roots in X-, Y- and Z-direction. Images of each time point were aligned using ImageJ Stackreg plugin (http://www.bigwww.epfl.ch/thevenaz/stackreg/

Web End =http://www.bigwww.ep.ch/thevenaz/stackreg/) before movie assembly.

Analysis of mitotic gure orientation. Seedlings were labelled with mouse anti-a-tubulin antibody (Santa Cruz). Z stacks were acquired from meristematic cells in prophase, metaphase and late cytokinesis, and images closest to the centre of the cell were selected. For each cell, length of the longitudinal cell edges and distances between the transversal cell edges and the points where the PPB ring or phragmoplast touched the longitudinal edges were measured. Deviation of PPB, mitotic spindle and phragmoplast orientation from the transversal central axis (midplane) of the cell was indicated by the absolute value derived by the following formula: 100 (distance between transversal edge and mitotic structure/total length of

longitudinal edge 200). A mean of 0% between the two values calculated for a cell

(one corresponding to each side) indicated a structure passing through the mid-plane, whereas 100% was one tracing a diagonal of the cell. Higher deviations occurred rarely (2.5% of 720 cells in total) and were considered as 100% for further analysis. Box-and-whisker plots were used to visualize deviation from the midplane in the 60 cells examined for each genotype and mitotic stage: for each data set they display median, upper quartile (blue box), lower quartile (red box), maximum (upper whisker) and minimum (lower whisker) of the population.

Live imaging of cortical microtubules. Cortical microtubules were analysed in epidermal cells of seedlings expressing mCherry-TUA5 (ref. 32) and AtEB1a-GFP35 or PIP5K3-YFP37. For movies of microtubule growth, Z stacks of planes intersecting the periclinal face of the cell 1 mm apart (0.51 mm in elongating wild-type cells) were imaged every 5 s for 2.5 min. Maximum intensity projections of each time point were used to assemble movies. For images of cortical microtubules in cells about to initiate outgrowth of a root hair, Z stacks of planes at 0.46 mm distance intersecting the periclinal face of the cell were acquired and employed to generate maximum intensity projections. Relative PIP5K3-YFP position was determined measuring the distance between basal trichoblast membrane and basal end of PIP5K3-YFP patch on the upper cell face and dividing it by total cell length. Measurements were performed on n 50 cells per genotype from

39 to 41 roots.

Microtubule orientation measurements. Seedlings with wild-type or sab-5 phenotype expressing the atrichoblast marker pGL2:NTF54 and mCherry-TUA5 (ref. 32) were imaged live. Trichoblasts were identied by absence of pGL2:NTF. Z stacks of planes at 0.45 mm distance were acquired on the periclinal face from either meristematic cells of 1318 mm or elongated cells of 2636 mm cell length. Maximum intensity projections were used to measure the angle of microtubules with respect to the root growth axis.

Generation of SAB-3xYpet and 3xYpet-SAB lines. A 3xYpet tag was fused to SAB at either the C- or the N-terminus by recombineering38. The JAtY clone 63C14, purchased from the John Innes Centre (Norwich, UK), was selected based on bacterial articial chromosome end sequence information from the Arabidopsis thaliana Integrated Database, http://www.atidb.org/cgi-perl/gbrowse/atibrowse/

Web End =http://www.atidb.org/cgi-perl/gbrowse/atibrowse/ . In this clone, the SAB-coding region is preceded and followed by 36 and 4.5 kb of gDNA, respectively. The JAtY transformation-competent bacterial articial chromosome was transferred to Escherichia coli strain SW105, kindly provided by Dr N. Copeland (Mouse Cancer Genetics Program, National Cancer Institute Frederick, Frederick, MD, USA)58. A recombineering cassette was generated by inserting the ampicillin resistance gene anked by ippase recognition sites at the 30-end of the codon-optimized 3xYpet tag38 (cassette available from the

NATURE COMMUNICATIONS | 4:2779 | DOI: 10.1038/ncomms3779 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 13

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3779

Arabidopsis Biological Resource Center, stock number CD3-1727). The cassette was amplied by PCR using two long recombineering primers (Supplementary Table S3): the forward primer included 50 nucleotoides homologous to the region upstream of the 3xYpet insertion point (until right after the rst codon of the SAB gene for the N-terminal fusion and before the STOP codon for the C-terminal fusion); the reverse primer included 50 nucleotides homologous to the region downstream of the insertion point (ending just after the rst codon or before the STOP codon of SAB, respectively). Cassette recombination into the JAtY clone was performed as described38, and the AmpR marker was removed on induction of the Flippase gene expression by arabinose. Constructs were sequenced and respective transformation-competent bacterial articial chromosome clones transformed into Agrobacterium tumefaciens GV3101, which was used to transform sab-5 heterozygous Arabidopsis plants by oral dipping38,59. Transformants were selected by Basta and further screened by PCR, ensuring presence of the sab-5-mutant allele, of the tagged SAB gene, and of the SacB gene (for primer sequences Supplementary Table S3). SacB lies close to the T-DNA left border and its presence conrmed transfer of at least one full copy of the T-DNA to the plant genome38. T2 seedlings were analysed for uorescence using a Zeiss LSM780 confocal microscope. Out of 12 SAB-3xYpet lines analysed, 7 displayed a very weak but identical uorescent signal. Out of 10 3xYpet-SAB lines, 9 showed the same pattern of uorescence, indistinguishable from SAB-3xYpet lines. One line was selected for each construct and T2 plants homozygous for the sab-5 allele and the SAB transgene were identied. Offspring of these plants was used for all further analyses and genetic crosses.

Plasmolysis. Plasmolysis was performed as described60. Seedlings were incubated in 1 ml of 0.8 M mannitol for 2 h under gentle agitation, stained for 30 s in10 mg ml 1 propidium iodide in 0.8 M mannitol, mounted on cover slips in the same medium and immediately imaged.

Generation of ER-ECFP-HDEL marker. The binary vector pGreenII0229 (ref. 61; obtained from the John Innes Centre, Norwich) was modied by adding a Cauliower mosaic virus 35S promoter (derived from the pSK36 vector62) and a Nopaline synthase terminator between the BamHI and SpeI, and at the NotI site of the multiple cloning site, respectively. The resulting vector was named p235S. ER signal peptide of Arabidopsis Basic chitinase (At3g12500) was amplied from gDNA using primers CHTTPFAscI and CHTTPRSpeI. PCR product was digested with AscI and SpeI and cloned into the corresponding sites of the p235S vector, generating the p235S-TP construct. Enhanced cyan uorescent protein (ECFP) coding sequence was amplied from the pECFP-C1 vector (Clontech) using primers CFPFSpeI and CFPERNotI, which introduced the coding sequence for the ER-retention signal peptide HDEL before the stop codon. PCR product was digested with SpeI and NotI and subcloned into the corresponding sites of the p235S-TP vector. After sequencing with 35SF and tNOSR primers, the resulting construct was transferred to A. tumefaciens strain C58C1 and used to transform Arabidopsis Col-0 plants by oral dipping59. T1 transgenic plants were selected by

Basta. Primer sequences are listed in Supplementary Table S3.

Visualization of SAB together with microtubules or F-actin. Seedlings expressing SAB-3xYpet or SAB-3xYpet and mCherry-TUA5 were xed for 40 min in PME buffer (50 mM piperazine-1,4-bis(2-ethanesulfonic acid), 2 mM MgSO4,

5 mM ethylene glycol-bis(b-aminoethylether)-N,N,N0,N0-tetraacetic acid, pH 6.9) supplemented with 0.1% Triton X-100 and 2% paraformaldehyde as described63. After three washes with PME buffer, seedlings were either mounted on cover slips in PME buffer and immediately imaged (for seedlings expressing SAB-3xYpet and mCherry-TUA5) or, for visualization of F-actin, mounted in 2% rhodamine phalloidin (Molecular Probes) in PME buffer and incubated 50 min in dark before imaging. To assess SAB-3xYpet localization relative to PPB, two sections (one through the middle of the cell and one 45 mm apart) were evaluated.

Lambda scan of root hair initiation sites. Lambda scans of trichoblasts undergoing hair initiation were captured from live Col-0 seedlings and seedlings expressing SAB-3xYpet, using a Zeiss LSM780 confocal microscope. On excitation at 514 nm, 32 images were acquired with narrow detection windows coveringthe spectral region between 416 nm and 686 nm, in Z stacks of planes at a distance of 2 mm. Maximum intensity Z projections were generated and lambda stacks were visualized in wavelength-coded colour view employing ZEN2010 (Zeiss) software.

Statistical analysis. Positioning of root hairs, ROP or PIP5K3-YFP patches along trichoblasts, and cell division and microtubule orientation angles did not follow a normal distribution. Thus, signicance of differences between distributions was tested with non-parametric, two-sample KolmogorovSmirnov test (http://www.physics.csbsju.edu/stats/KS-test.n.plot_form.html

Web End =http:// http://www.physics.csbsju.edu/stats/KS-test.n.plot_form.html

Web End =www.physics.csbsju.edu/stats/KS-test.n.plot_form.html ), which considers both distributions location and shape. Distributions of n 150 or 50 trichoblasts from

root hair and ROP or PIP5K3-YFP position measurements, respectively, of n 60

cells per mitotic stage, and of n 673802 or 578520 cortical microtubules were

analysed with signicance level Po0.05. Differences in trichome orientation were

analysed by F-test (Microsoft Excel), with n 174. Measurements of GFP-BASL

polar localization and trichome branching were analysed by Fishers exact test (http://www.quantitativeskills.com/sisa/statistics/fiveby2.htm

Web End =http://www.quantitativeskills.com/sisa/statistics/veby2.htm), with populations of n4187 and n4400, respectively, with signicance level Po0.05. Analysis of trichoblast length was performed by two-tailed, unpaired t-test with equal variance (computed with Microsoft Excel), with n 3 average values of independent

experiments and signicance level Po0.05.

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Acknowledgements

We are grateful to C. Ambrose, T. Aoyama, F. Berger, D.C. Bergmann, J.R. Ecker, D.W. Ehrhardt, N. Geldner, T. Hashimoto, S. Hennikoff, K. Palme, S. Persson,C.W. Lloyd, A. Sampathkumar, B. Scheres, K. Schumacher, S. Somerville andG.O. Wasteneys for sharing published materials and reagents. We thank the Nottingham Arabidopsis Stock Centre for providing seed stocks. This work was supported by Swedish Research Council (Vetenskapsrdet) Grant numbers 2006-522 and 2009-4846, and the Knut & Alice Wallenberg foundation project grant ShapeSystems to M.G., NSF Grant DBI0820755 to J.M.A. and NSF Grant MCB0923727 to J.M.A. and A.N.S.

Author contributions

M.G. conceived the project; M.G. and S.P. designed most of the experiments, S.P. performed the majority of the experiments; S.P., A.G., C.K., Y.I., L.K., P.H. and M.G. designed, performed and analysed experiments apart from recombineering work that was designed, performed and analysed by S.P., A.N.S. and J.M.A. M.G., S.P., A.G., C.K. andL.K. analysed and interpreted the results, A.N.S. and J.M.A. provided unpublished materials, valuable experimental advise, supervised and hosted S.P. during recombineering work. All authors contributed to the interpretation of results. M.G. and S.P. wrote the manuscript. All authors commented on and edited the manuscript.

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Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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Competing nancial interests: The authors declare no competing nancial interests.

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How to cite this article: Pietra, S. et al. Arabidopsis SABRE and CLASP interact to stabilize cell division plane orientation and planar polarity. Nat. Commun. 4:2779 doi: 10.1038/ncomms3779 (2013).

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/

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NATURE COMMUNICATIONS | 4:2779 | DOI: 10.1038/ncomms3779 | http://www.nature.com/naturecommunications

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The orientation of cell division and the coordination of cell polarity within the plane of the tissue layer (planar polarity) contribute to shape diverse multicellular organisms. The root of Arabidopsis thaliana displays regularly oriented cell divisions, cell elongation and planar polarity providing a plant model system to study these processes. Here we report that the SABRE protein, which shares similarity with proteins of unknown function throughout eukaryotes, has important roles in orienting cell division and planar polarity. SABRE localizes at the plasma membrane, endomembranes, mitotic spindle and cell plate. SABRE stabilizes the orientation of CLASP-labelled preprophase band microtubules predicting the cell division plane, and of cortical microtubules driving cell elongation. During planar polarity establishment, sabre is epistatic to clasp at directing polar membrane domains of Rho-of-plant GTPases. Our findings mechanistically link SABRE to CLASP-dependent microtubule organization, shedding new light on the function of SABRE-related proteins in eukaryotes.

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Title

Arabidopsis SABRE and CLASP interact to stabilize cell division plane orientation and planar polarity

Author

Pietra, Stefano; Gustavsson, Anna; Kiefer, Christian; Kalmbach, Lothar; Hörstedt, Per; Ikeda, Yoshihisa; Stepanova, Anna N; Alonso, Jose M; Grebe, Markus

Pages

2779

Publication year

2013

Publication date

Nov 2013

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms3779

ProQuest document ID

1458561180

Arabidopsis SABRE and CLASP interact to stabilize cell division plane orientation and planar polarity

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