the cowi locus of arabidopsis acts after rhdz, and in parallel with rhd3 and tipi, to determine

Plant Physiol. (1997) 115: 981-990

The COWI Locus of Arabidopsis Acts after RHDZ, and in Parallel with RHD3 and TIPI, to Determine the Shape, Rate

of Elongation, and Number of Root Hairs Produced from Each Site of Hair Formation'

Claire S . Crierson*, Keith Roberts, Kenneth A. Feldmann, and Liam Dolan IACR-Long Ashton Research Station, Department of Agricultura1 Sciences, University of Bristol, Long Ashton,

Bristol BSI 8 9AF, United Kingdom (C.S.G.); Department of Cell Biology, John lnnes Centre, Norwich NR4 7UH, United Kingdom (C.S.G., K.R., L.D.); and Department of Plant Sciences, Forbes Hall, University of Arizona,

Tucson, Arizona 85721 (K.A.F.)

Two recessive mutant alleles at CAN OF WORMSI (COWI), a new locus involved in root hair morphogenesis, have been identified in Arabidopsis fhaliana L. Heynh. Root hairs on Cowl - mutants are short and wide and occasionally formed as pairs at a single site of hair formation. l h e COWI locus maps to chromosome 4. Root hairs on Cowl- plants form in the usual positions, suggesting that the phenotype is not the result of abnormal positional signals. Root hairs on Cowl- roots begin hair formation normally, forming a small bulge, or root hair initiation site, of normal size and shape and in the usual position on the hair-forming cell. However, when Cowl- root hairs start to elongate by tip growth, abnormalities in the shape and elongation rate of the hairs become apparent. Ce- netic evidence from double-mutant analysis of cowl-1 and other loci involved in root hair development supports our conclusion that COW7 is required during root hair elongation.

~~

The morphogenesis of multicellular organisms is the result of coordinated changes in the number, position, size, and shape of cells. Cell shape has two different roles: First, coordinated changes in cell shape are required during the elaboration of multicellular structures; for example, during anther maturation, coordinated changes in the elongation of a subset of cells produce the anther stalk. Second, cell shape is important for the function of a number of special- ized cell types; for example, leaf trichomes have a polarized structure and shape critica1 to their function in protecting against grazing by insects. Molecular genetic approaches have revealed mechanisms governing cellular morphogenesis, the process of shape change, in Drosophila melanogaster (Adler, 1992; Wong and Adler, 1993) and yeast (Kron and Gow, 1995). These approaches are now being applied to

This work was supported by Biotechnology and Biological Science Research Council Stem Cell Molecular Biology grant no. AT 208/583 and by a Royal Society Dorothy Hodgkin Research Fellowship awarded to C.G. The John Innes Centre and IACR receive grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom. This is institu- tion paper no. 34,267.

*. Corresponding author; e-mail [email protected]; fax 44-1275-394281.

plant cells, particularly leaf trichomes and root hairs. Whereas 18 loci have been implicated in the processes of trichome morphogenesis (Hülskamp et al., 1994), only 6 involved in root hair morphogenesis have been described (Schiefelbein and Somerville, 1990; Schiefelbein et al., 1993, Masucci and Schiefelbein, 1994). We have identified a new locus involved in root hair morphogenesis, C A N OF WORMSZ (COWZ).

Root hairs are long, tubular outgrowths on the surface of plant roots. They are thought to be important for the up- take of nutrients and water and are the site of entry for Xkizobium bacteria on nodulating plants. Each root hair is formed by a single cell, usually in the epidermis. These cells are produced by divisions in the root meristem (Dolan et al., 1993) and, along with other cells in the root, elongate and expand radially. As elongation is ceasing, hair-forming cells undergo an extreme change in cell shape: growth becomes targeted to a very small area on the outer cell surface and a long, thin tube is formed.

The main part of the root hair forms by tip growth. During tip growth the shape of the cell is determined by the precise targeting of vesicles carrying cell wall precur- sors to the growing tip. Tip growth is a mechanism of morphogenesis common to many cell types. Important ex- amples of tip-growing structures include pollen tubes and funga1 hyphae.

Root hairs of Arabidopsis thaliana L. Heynh are very ame- nable to study. As in other plants, Arabidopsis root hairs form on the surface of the plant and their development is readily observed. Viable mutants are easy to isolate and molecular genetic tools are available, making it relatively easy to isolate genes identified by mutation (Schiefelbein and Somerville, 1990). Arabidopsis root hairs form in a pattern of rows of hair-forming cells interspersed with rows of non-hair-forming cells (Dolan et al., 1994). Conse- quently, it is possible to predict which cells are going to form hairs and to observe their development from a very early stage, before the hair starts to develop.

Abbreviations: CAPS, cleaved amplified polymorphic sequence; EMS, ethyl methanesulfonate.

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Mechanisms governing the patterning of root hair cells have recently been identified. Root hair formation is stim- ulated by the plant-growth regulators ethylene and auxin. Non-hair-forming cells are apparently prevented from re- sponding to these signals by the putative transcriptional regulator TTG and the transcription factor GL2 (Dolan et al., 1994; Galway et al., 1994; Masucci and Schiefelbein, 1994, 1996; Tanimoto et al., 1995; Di Cristina et al., 1996; Masucci et al., 1996).

To aid in the classification of root hair mutants and in the description of root hair development, we have divided root hair formation into three phases. In phase 1, a small bulge forms at the end of the cell nearest to the root tip. In phase 2, slow tip growth occurs from a region of this bulge. In phase 3, the remainder of the hair forms by rapid tip growth. The transition from phase 2 to phase 3 is variable and takes place when the hair is between 20 and 40 pm long (Dolan et al., 1994). During phase 1 of root hair formation, the positioning of the root hair initiation site is affected by the plant growth regulators auxin and ethylene (Masucci and Schiefelbein, 1994). Roots treated with chem- icals that block either ethylene synthesis (aminovinyl Gly) or the perception of ethylene (Ag+) produce root hairs at a more apical position (near the shoot). The application of the ethylene precursor ACC to roots results in the formation of initiation sites in a more basal (near the root tip) position.

Mutations in the RHD6 gene result in the development of fewer hairs, and hairs that do form occupy a more apical location, indicating that this gene is involved in establish- ing the initiation site (Masucci and Schiefelbein, 1994). The formation of the initiation site during phase 1 is thought to depend on local loosening of the cell wall. Mutations in the RHDl gene result in hairs with bulbous bases. This phenotype has been interpreted as indicating that the RHDl gene product normally regulates the degree of loosening of the cell wall during root hair initiation (Schiefelbein and Som- erville, 1990).

Once a small bulge has been established at the root hair initiation site, it begins to elongate by slow tip growth (phase 2). Very little is known about the mechanisms involved at this stage of root hair development; studies of root hair elongation have usually been conducted using longer hairs. Mutations at the root hair development locus RHD2 result in the formation of bulges that do not elongate (Schiefelbein and Somerville, 1990), but this phenotype has not been described further. Roots treated with ABA produce short, bulbous root hairs; it is not known at which stage of root hair morphogenesis these hairs are affected (Schnall and Quatrano, 1992).

In phase 3 root hairs elongate by rapid tip growth. Dur- ing phase 3 the shape of the hair is the result of the precise targeting of vesicles carrying new cell wall material to the growing tip. As in other tip-growing cells, this targeting is thought to be orchestrated by the interaction of actin- mediated vesicle transport with a tip-high Ca2+ gradient (Reiss and Herth, 1979; Kohno and Shimmen, 1987, 1988a, 1988b; Schiefelbein et al., 1992; Herrmann and Felle, 1995). The disruption of microtubules, actin microfilaments, or the Ca2+ gradient inhibits tip growth (Lloyd et al., 1987;

Heslop-Harrison et al., 1991; Alfano et al., 1993). There is evidence for a role of protein phosphatases in root hair development; application of two inhibitors of Ser / Thr protein phosphatases has been shown to potently inhibit both the initiation and elongation of root hairs in Arabidopsis (Smith et al., 1994).

Mutations in the RHD3, RHD4, and TlPl genes result in the production of abnormal root hairs. In all three cases, root hairs are apparently produced in normal positions on the hair-forming cell, suggesting that these mutations do not affect hair initiation (phase 1; Schiefelbein and Somer- ville, 1990; Schiefelbein et al., 1993). The RHD3 gene encodes a protein with GTP-binding motifs that is required during vacuole enlargement and tip growth (Galway et al., 1997; Wang et al., 1997). tipl mutants have defective pollen tubes and root hairs, suggesting that the TlPl gene is involved in a process common to tip growth in root hairs and pollen tubes (Schiefelbein et al., 1993). It is not clear whether TIPl is required for tip growth during phase 2 or phase 3 of root hair elongation. There is genetic evidence that the plant growth regulators auxin and ethylene are involved in root hair elongation. Mutants at the A X R l (Cernac et al., 1997), AXR2 (Wilson et al., 1990), AXR3 (Leyser et al., 1996), and AUXZ (M. Estelle, personal communication) loci are auxin-resistant and have short root hairs. Dominant mutations in the ETRZ ethylene receptor that render plants insensitive to ethylene also result in short root hairs (M. Estelle, personal ,communication).

Mutations at the COWl locus affect the shape, elongation rate, and number of root hairs formed at each initiation site. Apart from the root hair phenotype, Cowl- plants have a normal growth habit and are fully fertile, suggesting that the defect is specific to root hairs. Phenotypic characterization of root hairs of Cowl- mutants indicates that COWl is not required during phase 1 of hair formation (initiation) but has an important role during phases 2 and 3 (elongation). This conclusion is supported by genetic evidence from double mutant phenotypes.

MATERIALS A N D METHODS

Genetic Lines

cowl-1 was isolated from a T-DNA-mutagenized population of the WS ecotype of Arabidopsis thaliana L. Heynh (Feldmann and Marks, 1987; Feldmann et al., 1989). cowl-1 lines isolated after two rounds of outcrossing to WS were used for experiments. cowl-2 was isolated from the M, seeds of plants raised from A . thaliana ecotype Columbia seed (M,) treated with 0.4% EMS in 100 mM sodium phos- phate buffer for 8 h. cowl-2 lines isolated after two rounds of outcrossing to ecotype Columbia were used for experiments. The Landsberg erecta ecotype and the WlOO multiple marker line were obtained from the Nottingham Ara- bidopsis Stock Centre (UK). Seeds of rkdl, rkd2, rhd3, rhd4, tipl, and rhd6 were the kind gift of John Schiefelbein.

Growth Conditions

Seedlings were grown on Murashige-Skoog medium (Flow Laboratories, Irvine, Scotland, UK) containing 3%

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Role of Arabidopsis COW7 Locus in Root Hair Morphogenesis 983

Suc and agarose (1% unless otherwise stated). Seeds were germinated with the plates in a vertical orientation at 22°C with constant illumination. Plants were transferred at the eight-leaf stage to compost supplemented with slow- release fertilizer (Osmocote, Sierra United Kingdom, Not- tingham) and grown for 6 to 8 weeks until seeds could be collected.

Genetic Mapping of COWl

WlOO (Koornneef et al., 1987) plants were pollinated using cowl-l pollen and the progeny were allowed to self-fertilize to produce F, seeds. A total of 969 F, seedlings grown in Petri plates were scored for the cowl-l phenotype, transferred to compost, and allowed to grow to ma- turity. Each was scored for the 10 markers carried by the WlOO parental line. The data were processed using Map- maker 3.0 and the Kosambi function.

F, progeny from a cross between cowl-1 (WS background) and Landsberg erecta were grown to the eight-leaf stage and genomic DNA from each individual was pre- pared according to the method of Dellaporta (1994). CAPS primers (Research Genetics, Huntsville, AL) were used as described by Konieczny and Ausubel (1993). The results were analyzed using Mapmaker 3.0 (Lander et al., 1987; Lincoln et al., 1992) and the Kosambi function.

Cryoscanning Electron Microscopy

Five-day-old seedlings were frozen in N, slush at -190°C. Ice was sublimed at -70°C and the specimen sputter-coated with gold before being examined on a scan- ning electron microscope (model 505, Philips) fitted with a cold stage.

Light Microscopy

Dark- Field Microscopy

Seedlings were observed and photographed using dark- field illumination and a stereozoom microscope (model MZ6, Leica).

Counting Twin Hairs

Five-day-old plants were transferred to a microscope slide and a drop of Murashige-Skoog medium containing 3% SUC was applied. Root hairs were viewed using differential interference optics on an Axiophot microscope (Zeiss).

Measuring Root Hairs

Five-day-old plants growing in Petri dishes were placed on the stage of a microscope (model Microphot SA, Nikon) and the region of the root where root hairs ceased elongating and reached a uniform maximum length was identified. Root hairs in this region were imaged using differen- tia1 interference optics and long-distance working objectives and relayed to a video monitor using a video

camera (model TK-l280E, JVC, Tokyo, Japan). Root hair shapes were traced onto cellophane placed over the monitor and the tracings were measured later.

Differential lnterference Microscopy of Developing Root Hairs

For Figure 3, A and C, 4- and 5-d-old plants were grown on thin layers of Murashige-Skoog medium containing 3% Suc and 0.8% agarose, so that the roots grew within the solid medium. Seedlings were transferred to a microscope slide with their roots still embedded in solid medium, a drop of Murashige-Skoog medium containing 3% SUC was added, and a coverslip supported by petroleum jelly was overlaid. Root hairs were observed using differential interference optics on an Axiophot microscope. Only root hairs that showed cytoplasmic streaming were photographed.

For Figure 3B, 2-d-old seedlings were transferred to fresh, thin layers of Murashige-Skoog medium (Flow Lab- oratories) containing 3% SUC and 1% agarose. A coverslip supported by petroleum jelly was carefully overlaid so that moisture was trapped around the root. Between observations slides were incubated in a humid environment at room temperature in the dark. Photographs were taken using a DMRB microscope (Leica).

Fixation, Embedding, and Sectioning

Five-day-old seedlings were fixed in ice-cold 2.5% glu- taraldehyde in 50 mM cacodylate buffer, pH 7, for 1 h, rinsed twice in ice-cold water for 5 min, and dehydrated in 70 and 90% ethanol for 10 min each. Seedlings were infil- trated for 10 min in 50% Historesin (Reichert-Jung, Vienna, Austria) in 95% ethanol and for 30 min in 100% Historesin before being polymerized at room temperature overnight. Sequential 3-km seria1 sections were cut with a dry glass knife using an ultrotome (model 4801A, LKB, Bromma, Sweden), floated on water, and dried onto glass slides. Tissue was stained with methylene bluelazure A for 10 min, washed, and viewed using an Axiophot microscope.

Time-Lapse Measurements of Root Hair Growth

ried out as described by Dolan et al. (1994). Time-lapse measurements of root hair growth were car-

Double-Mutant Analysis

cowl-1 was crossed with other root hair mutants and the F, progeny were selfed to produce F, populations. F, progeny that displayed only one of the parental phenotypes were selfed to produce F, seeds. A proportion of the F, families segregated for the double-mutant phenotype. Mul- tiple F, families segregating for each double-mutant phenotype were identified. Plants with double-mutant phenotypes were crossed to the wild type, and F, progeny were examined to confirm that both parental phenotypes could be recovered.

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RESULTS

Isolation of cowl Mutants from T-DNA- andEMS-Mutagenized Arabidopsis Lines

Figure 1 shows roots and root hairs of wild-type, cowl-l,and cowl-2 Arabidopsis seedlings. The cowl-l allele wasisolated from a population of 7000 T-DNA-mutagenizedlines (Feldmann and Marks, 1987; Feldmann et al., 1989).

Figure 1. Root tips of 4-d-old seedlings of wild type (ecotype Co-lumbia), cowl-l, and cowl-2.

The cowl-2 allele was isolated in a screen of 75,000 seeds(M2) from 6000 lines of an EMS-mutagenized population(M,). Root hairs on cou'1-1 and cowl-2 plants were shorterand wider than normal (Fig. 1) and their appearance wasreminiscent of nematode worms, so the locus was namedCAN OF WORMS 1. No differences in the growth habits orfertility between cowl-l or cowl-2 plants and wild-typeplants were observed.

Data from crosses of cowl-l and cowl-2 with wild typeare given in Table I. The results demonstrate that cowl-land cowl-2 are recessive, nuclear, single-locus mutations.The progeny of crosses between cowl-l and cowl-2 hadmutant root hairs (Table I), indicating that these two linescarry alleles of the same gene. F, progeny of crosses be-tween cowl-l and the previously reported root hair mu-tants rhdl to rhd4, rhd6, and tipl were wild type, demon-strating that COW1 is a new gene in the root hairdevelopment pathway.

The cowl-l mutation was mapped relative to visiblemarkers by scoring an F, population from crosses betweencowl-l and the multiple marker line W100 (Koornneef etal., 1987) and relative to molecular markers by scoring theF2 progeny from a cross between cowl-l (WS background)and Landsberg erccta for CAPS markers, cowl-l mapped tochromosome 4, 13 centiMorgans from cer2 and 9 centiMor-gans from the CAPS marker PG11.

Characteristic Features of cow/-/ andcowl-2 Phenotypes

Cryoscanning electron micrographs shown in Figure 2reveal some pairs of hairs emerging from the same site onCowl~ roots (Fig. 2, C and D). We have called these "twin"hairs. Twin hairs appeared in the same cell files as singlehairs.

The mean dimensions of full-grown root hairs on wild-type Columbia and cowl-2 roots are shown in Figure 2E.cowl-2 single hairs were one-quarter the length and 2.5times the diameter of wild-type hairs, cowl-2 twin roothairs were about the same length as cowl-2 single hairs (i.e.about one-quarter the length of the wild type). However,cowl-2 twin hairs were narrower than single cowl-2 hairs,suggesting that twin hairs are not simply pairs of mutantsingle hairs.

Root hair formation in Arabidopsis is known to be de-termined by positional information (Dolan et al., 1994);root hairs are formed only by epidermal cells overlyinganticlinal walls between underlying cortical cells. To inves-tigate whether twin hairs might be produced in response toabnormal positional signals, the anatomy of cowl-2 rootswas investigated. Figure 2F shows transverse sections ofwild-type and cowl-2 roots. Cells bearing twin hairs werelocated in the same position as wild-type root hairs, sug-gesting that the twin hair phenotype is not the result ofabnormal positional signals.

cowl roots produced twin hairs more frequently thanwild-type roots (Table II). Heteroallelic cowl-llcowl-2plants produced twin hairs at 30% of hair initiation sites,compared with 1.9% for cowl-l, 17% for cowl-2, and 0.24%for ecotype Columbia. The large proportion of twin hairs

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Role of Arabidopsis COW7 Locus in Root Hair Morphogenesis 985

Table 1. Genetic analysis of Cowl - plants Root Hairs

Wild t w e Short, fat Cross Type Total X 2

COWl/COWl X COWl- l /COWl- l (T-DNA) F, 156 156 O

CO Wl/CO W l X co w 1 -~/co w 1-2 (E MS) F, 78 78 O

cow 1 - l k o w 1-1 x cow 1 -2kow 1-2 F, 91 O 91

F2 62 1 451 170 1.87

F, 1995 1487 508 0.23

a x2 calculated for a 3:l ratio of wild-tvpe phenotvpe to mutant phenotvpe, P 2 0.05 when x2 5 3.84.

on heteroallelic roots is remarkable; if there were no interaction between alleles, we would expect to see an interme- diate phenotype between cowl-1 and cowl-2 in the heteroallelic plants. In fact, we see a significantly higher frequency of twin hairs on heteroallelic roots. This interaction between cowl alleles suggests that the COWl gene product may function as a multimer. However, the cowl-1 and cowl-2 lines have different wild-type backgrounds (WS and Columbia, respectively) and we cannot rule out the possibility that the hybrid nature of heteroallelic plants is responsible for the phenotype.

We have noted that orientation of twin hairs is a distinc- tive feature of Cowl- roots. The majority of twin hairs lie across the root, perpendicular to the long axis of the hair- forming cell (Table 111).

Cowl - Root Hairs Are Defective in Root Hair Elongation

We previously described three phases of root hair formation. In the first phase, a small bulge appears at the basal end of the hair-forming cell. In the second phase, a slow tip-growing hair develops from this bulge. In the third phase, the rate of tip growth is increased. The transition from phase 2 to phase 3 is variable and takes place when the hair is between 20 and 40 pm long (Dolan et al., 1994).

Comparison of wild-type, cowl-1, and cowl-2 roots (Fig. 1) shows young root hairs at a similar distance from the root tip in a11 three cases. This suggests that phase 1 of root hair formation is occurring at the same stage of epidermal development on wild-type, cowl-1, and cowl-2 plants. Fig- ure 3A shows differential interference contrast images of young root hairs on wild-type, cowl-1, and cowl-2 roots. Bulges on cowl-1 and cowl-2 roots were in the usual position, at the end of the cell nearest the root tip (Fig. 3A), suggesting that Cowl- root hairs can undergo phase 1 successfully .

Figure 38 shows root hairs at early (in phase 2 or very early in phase 3 of development, between 16 and 28 pm long) and late (phase 3) stages of development on wild- type and Cowl- roots. The same root hairs were photographed at each stage. Wild-type root hairs at an early stage of development had a conical shape, tapering slightly toward the tip. At later stages wild-type root hairs were narrower along most of their length than at the base (Figs. 2E and 3B). Cowl- root hairs at an early stage of development did not taper toward the tip as much as did wild-type hairs. At later stages, when wild-type root hairs were narrower along most of their length than at the base, Cowl-

single hairs were a similar diameter along their length as at the base (Figs. 2E and 38). No differences were observed between the size or shape of young root hairs on Cowl- roots that went on to form single mutant hairs and those that went on to form twin hairs. Figure 3C shows high- resolution differential interference contrast images of twin hairs between 30 and 35 pm long (in late phase 2 or early phase 3) on cowl-2 roots. Twin hair branches can be seen initiating on these hairs. Therefore, twin hair branches initiate late (phase 2 or phase 3) in hair development. Both branches initiate at about the same time.

Elongation rates of wild-type and cowl-1 root hairs during phases 2 and 3 were observed using time-lapse video microscopy, and early and late elongation rates were determined as previously described (Dolan et al., 1994). The results revealed that cowl-1 root hairs elongate slowly during both phases 2 and 3 (Fig. 4; Table IV). Wild-type root hairs elongated at 0.36 to 0.42 pm min-l during phase 2 and then more rapidly, at 0.9 to 1.22 pm min-', during phase 3. cowl-1 root hairs elongated at 0.14 to 0.17 pm min-' during phase 2, and in phase 3 (when wild-type hairs elongated more quickly), cowl-l root hairs elongated at the same rate or more slowly than in phase 2, at 0.07 to 0.2 pm min-l.

COW7 Acts between RHD2 and RHD4, and in Parallel with RHD3 and TW7, during Root Hair Development

Double mutants carrying cowl-2 and other root hair mutations (Fig. 5, A-H) were used to determine when COWl acts during root hair development. rkd6 and rkd2 were epistatic to cowl-1, demonstrating that COWl acts after the RHD6 and RHD2 genes during root hair development. r k d l cowl-1, tipl cowl-1, and rhd3 cowl-1 double mutants had additive phenotypes, suggesting that COWl functions in parallel pathways with RHD1, TIP1, and RHD3. cowl-1 was epistatic to rkd4, indicating that COWl acts before RHD4 during root hair development. The results place COWl between RHD2 and RHD4, and in parallel with RHD3 and TIPl in the genetic pathway of root hair formation (Fig. 51).

D I SC U SSI ON

COW7 1s a New Locus Required for Normal Root Hair Formation

We have identified a new locus, COWl . Two recessive alleles, cow-1 and cowl-2, have been characterized genet-

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1139 ±168

Wildtype 19.1+2, 7.6 ± 0.7

20.3 ± 2.5

COW1-2 20.9 + 2.!-266 ± 27

COW1-2 35.7 + 3.3

Figure 2. Cow! ~" root hairs are short and wide and are sometimes formed as pairs (twin hairs) at the same site. Scanningelectron micrographs of developing root hairs of wild-type (WS; A), cow7-7/cow7-7 (B), cow1-2/cow1-2 (C), andcow7-7/cow7-2(D). In C and D, single root hairs can be seen in the same files as twin hairs (O). E, Dimensions of wild-typeroot hairs, cow7-2 single root hairs, and cow1-2 twin root hairs. Values (in micrometers) are the means ± so of between5 and 22 measurements. F, Transverse sections of 4-d-old roots of wild-type (wt; ecotype Columbia) and cowl-2 plants.Twin hairs form overlying the anticlinal wall between underlying cortical cells. Bars, A to D, 100 jam; F, 25 ju,m.

ically and morphologically. The root hairs of cowl-1 andcowl-2 plants are shorter and wider than the wild type.Hairs on cowl-1 and cowl-2 roots occasionally form inpairs (twin hairs) from the same site. A similar combinationof phenotypes has been observed in tipl plants, which haveshort root hairs that branch at the base to produce two or

three hairs from a single site (Schiefelbein et al., 1993). Inaddition, tipl plants have defective pollen tube growth,and, consequently, tipl is thought to affect tip-growthmechanisms common to both root hairs and pollen tubes(Schiefelbein et al., 1993). Unlike tipl, cowl-1 and cowl-2plants are fully fertile and do not display distorted segre-

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Role of Arabidopsis COW/ Locus in Root Hair Morphogenesis 987

Table II. Frequency of twin hair formation by wild-type andCow1~ plants

Genotype

ColumbiaWScow 1-1 /cowl -1cow1-2/cow1-2cow1-1/cow1-2

RootsExamined

1010101210

SingleHairs

no.416480519328451

TwinHairs

10

1067

195

TwinHairs

%0.2401.9

17.030.0

gation, suggesting that pollen tube growth is not affected.As far as we can tell from phenotypic and anatomicalcharacterization of mutant plants, mutations at the COW1locus affect only root hairs.

COW1 Is Required for Root Hair Elongation

By comparing observations of wild-type root hairs withthose of Cowl ~ root hairs, we have been able to pinpointwhere the development of Cowl ~ root hairs diverges fromthe wild type and thereby determine the stage at which theCOW1 gene acts.

Our results suggest that the COW2 gene is not requiredfor phase 1 of root hair formation but contributes to bothroot hair shape and elongation rate in phases 2 and 3. Phase1 appears to be normal in cowl-1 and cowl-2 plants; roothairs begin to form in the same region of the root and,therefore, probably at the same stage of epidermal devel-opment as in the wild type and are in the same position onthe surface of the hair-forming cell as wild-type hairs. Thedefect in Cowl" hairs affects elongating root hairs inphases 2 and 3. During phase 2 the elongation rate ofcowl-1 root hairs is slower than in wild type. At this stagethere is a slight difference in the appearance of wild-typeand Cowl" root hairs; wild-type hairs are slightly moretapered toward the tip. The effects of defects in the COW1gene are more noticeable at the transition between phase 2and phase 3. At this stage, differences in the shape ofwild-type and Cowl" root hairs become much stronger,and twin-hair branches appear on a proportion of Cowl ~root hairs. In phase 3, when the elongation rate of wild-type root hairs increases, the elongation rate of cowl-1 roothairs stays the same or decreases.

The conclusion that the COW1 gene does not affect hairinitiation, but is required during root hair elongation, issupported by genetic evidence from double mutants con-structed with other mutants that affect these two processes.RHD6 is required during root hair initiation (Masucci and

Table III. Orientation of twin

Genotype

cow1-1/cow1-1cow1-2/cow1-2cowl-1 /cowl -2

RootsExamined

101210

hairs formed by Cowl plantsTwin

Hairs Lyingalong Root

no.

12542

TwinHairs Lyingacross Root

942

152

TwinHairs Lyingacross Root

%

906378

wt cowl-1 cowl-2

B early late

Figure 3. Cowl ~ root hairs are defective in root hair elongation. A,Very young root hairs on wild-type (wt, Columbia), cowl-1, andcowl-2 roots. B, Root hairs photographed at early and late stages ofroot hair development on wild-type (wt, Columbia x WS F,) andCowl" (mt, cow/-/ cowl-2) roots. The same root hairs are shownat each stage. Arrows indicate twin hairs. C, Young twin hairs oncow1-2 roots. Arrows indicate nascent branches on each twin hair.Bars, A and C, 20 /urn; B, 50 /j.m.

Schiefelbein, 1994), and RHD2 is required later for root hairelongation (Schiefelbein and Somerville, 1990). rhd6 and

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500-

400

£ 300-oic4>

200-

oo100-

0

cow1-1 acowl-1 bcowl-1 cColumbia aColumbia bWS

' D B 8 %

0 2 0 0 4 0 0 6 0 0 8 0 0

time (min)Figure 4. cowl-1 root hairs elongate slowly.

r/K/3caw1-1

rhd4 rhd4 rhd€ caw1-1 tipl tiplcowl-1

rhd2 are epistatic to cowl-1, confirming that the COW1gene acts later in the sequence of root hair developmentthan these two genes.

RHD3 and RHD4 act sequentially during root hair elon-gation (Schiefelbein and Somerville, 1990), and T1P1 isthought to be required for tip growth (Schiefelbein et al.,1993). rhd3 cowl-1 and tipl cowl-1 double mutants haveadditive phenotypes, suggesting that the COW1 gene op-erates in parallel with RHD3 and TIPl. This indicates thatroot hair elongation involves multiple genetic pathwaysthat can operate independently of each other, cowl-1 isepistatic to rhd4, indicating that RHD4 acts later in root hairdevelopment than COWl. Like rhdl rhd2, rhdl rhd3, andrhdl rhd4 double mutants (Schiefelbein and Somerville,1990), rhdl cowl-1 double mutants have an additive phe-notype, confirming that, along with these other root hairdevelopment genes, COW1 acts in a pathway parallel toRHDl. When combined with previously published infor-mation (Schiefelbein and Somerville, 1990; Schiefelbein etal., 1993; Masucci and Schiefelbein, 1994), our double mu-tant results indicate that COW1 is part of a root hair devel-opment pathway that begins with RHD6 and ends withRHD4 and that COW1 is a member of a separate root hairelongation pathway from TIPl or RHD3.

Table IV. Elongation rates of wild-type and cowl-1 root hairs

GenotypeElongation Rate

Early Late

ColumbiaColumbiaWScow/-/cow/-/cow/-/

0.360.380.420.140.170.16

/Am min '

1.221.210.900.080.070.20

covwRHD6 -^~ RHD2 RHD4

RHD3TIP1

RHD1

Figure 5. Root hair phenotypes of double mutants. A, Roots of4-d-old seedlings of rhdl cowl-1 double mutant displaying thecharacteristic rhdl phenotype with bulges on epidermal cells partic-ularly noticeable at the root tip. As previously described (Schiefel-bein and Somerville, 1990), rhdl hair-forming cells often form hairs,but in the rhdl cowl-1 double mutant these hairs have a cow1-lphenotype. B, rhd2 COWl. C, Left, rhd2 cowl-1 double mutant;right, RHD2 cowl-1. The rhd2 cow/-/ double mutant has an rhd2phenotype (compare with rhd2 root in B), forming root-hair initiationsites, but very few hairs. D, Left, rhd3 COWl; right, rhd3 cowl-1double mutant. The rhd3 COWl root has characteristic fine, wavyroot hairs (Schiefelbein and Somerville 1990). The rhd3 cowl-1double mutant has very short, fat root hairs, which are shorter thanroot hairs on cowl-1 single-mutant plants (compare with cowl-1roots in C and F). E, Left, rhd4 COWl; right, rhd4 cowl-1 doublemutant. rhd4 cowl-1 double-mutant root hairs have the same phe-notype as RHD4 cowl-1 hairs (compare with cowl-1 roots in C andF). F, Left, rhd6 cowl-1 double mutant; right, RHD6 cowl-1. rhd6cowl-1 roots had the same phenotype as r/Jc/6 roots (Masucci andSchiefelbein 1994), producing very few root hairs on the main part ofthe primary root. C, tipl cowl-1 double mutant. The tipl cowl-1double mutant has very short, fat root hairs, which are shorter thanroot hairs on cowl-1 single-mutant plants (compare with cowl-1roots in C and F). H, tipl COWl root with characteristic root hairs,which are longer and thinner than those of the tipl cowl-1 doublemutant in C. I, Genetic pathway of root hair formation implied bydouble-mutant results and previously published data.

The results of morphological and genetic characteriza-tion of mutants in the COWl gene led us to conclude that

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Role of Arabidopsis COWl Locus in Root Hair Morphogenesis 989

COWl is required during root hair elongation, which occurs by tip growth. A number of treatments that affect tip growth have been reported, including treatments that inhibit phosphatases or disrupt microtubules, actin microfilaments, or Ca2+ gradients. Only one of these has effects similar to mutations in the COWl gene. Funga1 mycelia grown on very low concentrations of free Ca2+ are more highly branched than normal, the hyphae are twice the normal diameter, and the hyphal elongation rate is reduced 8-fold (Robson et al., 1991).

These changes in hyphal growth resemble the changes in root hair growth that we see in Cowl- plants, in which root hairs are branched at the base. and have between 1.8 and 2.7 times the normal diameter and the root hair elongation rate is reduced between 4.5- and 13-fold. However, studies of Arabidopsis root hairs cast doubt on the idea that the function of COWZ could be related to Ca2+. Al- though growth on low concentrations of free Ca2+ reduced the length of Arabidopsis root hairs, no effects on hair diameter or branching were reported (Schiefelbein et al., 1992). An understanding of the mechanism of COWl action awaits the molecular and biochemical characterization of the gene. cowl-1 plants contain a single kanamycin- resistance locus that co-segregates with the mutant phenotype (data not shown). Work to isolate the COWl gene is in progress.

ACKNOWLEDCMENTS

We are grateful to John Schiefelbein for the gift of seeds of rhdl-rhd4, rhd6, and tipZ and to P. Scolnick (DuPont, Wilmington, DE) for his hospitality and advice. We would also like to thank Katharina Schneider for assisting with the isolation of the cowl-2 allele and Mike Holdsworth for performing some of the crosses. The Landsberg erecta ecotype and WlOO mapping line were grate- fully received from the Nottingham Arabidopsis Stock Centre. Claire Grierson would like to acknowledge the support of the biometric, photographic, microscopic, and horticultural staff at IACR-Long Ashton and to thank Jill Parker for valued assistance. We also thank R. Scott Poethig for support and suggestions.

Received May 23, 1997; accepted July 29, 1997. Copyright Clearance Center: 0032-0889/97/115/0981/10.

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the cowi locus of arabidopsis acts after rhdz, and in parallel with rhd3 and tipi, to determine

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