The Plant Cell, Vol. 14, 1017–1031, May 2002, www.plantcell.org © 2002 American Society of Plant Biologists

hydra

Mutants of Arabidopsis Are Defective in Sterol Profiles and Auxin and Ethylene Signaling

Martin Souter,

a,1

Jennifer Topping,

a,1

Margaret Pullen,

a

Jiri Friml,

b

Klaus Palme,

b

Rachel Hackett,

c

Don Grierson,

c

and Keith Lindsey

a,2

a

Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, University of Durham, South Road, Durham DH1 3LE, United Kingdom

b

Max-Delbrück Laboratorium, Carl von Linne Weg 10, D-50829 Köln, Germany

c

Plant Science Division, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, United Kingdom

The

hydra

mutants of Arabidopsis are characterized by a pleiotropic phenotype that shows defective embryonic andseedling cell patterning, morphogenesis, and root growth. We demonstrate that the

HYDRA1

gene encodes a

�

8-

�

7sterol isomerase, whereas

HYDRA2

encodes a sterol C14 reductase, previously identified as the

FACKEL

gene prod-uct. Seedlings mutant for each gene are similarly defective in the concentrations of the three major Arabidopsis ste-rols. Promoter::reporter gene analysis showed misexpression of the auxin-regulated

DR5

and

ACS1

promoters and ofthe epidermal cell file–specific

GL2

promoter in the mutants. The mutants exhibit enhanced responses to auxin. Thephenotypes can be rescued partially by inhibition of auxin and ethylene signaling but not by exogenous sterols orbrassinosteroids. We propose a model in which correct sterol profiles are required for regulated auxin and ethylenesignaling through effects on membrane function.

INTRODUCTION

Sterols are essential components of fungal, plant, and ani-mal membranes. They regulate fluidity and interact with lip-ids and proteins within the membrane, and they are theprecursors for the brassinosteroid (BR) hormones in plants(Hartmann, 1998). The sterol biosynthetic pathway in plants,therefore, can be viewed as comprising two parts: onebranch produces the bulk membrane sterols (the principalsterols in Arabidopsis being stigmasterol, campesterol, andsitosterol), and the second part represents the BR synthesisbranch. Sterol biosynthesis has been well characterized inyeast, supported by a powerful system of genetic analysis.In animals, and more recently in plants, sterol biosyntheticenzyme function has been confirmed via the functionalcomplementation of yeast mutants (Gachotte et al., 1996).Functional analysis of sterol function in plants has involved arange of approaches, but recently, genetic studies haveprovided useful information on the requirement for particularenzymes in sterol and BR biosynthesis and, for BRs, per-ception and signal transduction (Clouse, 2000; Diener et al.,2000; Schaeffer et al., 2001).

In animals, sterols appear to be important to maintain cor-rect cell-signaling activities. For example, drugs such as the

�

ligand SR31747A, which inhibits the activity of the

�

re-ceptor (emopamil binding protein [EBP], which has

�

8-

�

7sterol isomerase activity), cause defects in a diversity of cel-lular processes, including the inhibition of mammalian lym-phocyte proliferation in response to mitogens (Derocq et al.,1995) and the inhibition of graft rejection in mouse via themodulation of gene expression (Carayon et al., 1995), andthey may influence lipoprotein functions leading to immuno-suppressive effects (Dussossoy et al., 1999). In plants, alack of detailed pharmacological studies has precludedanalogous investigations of the role of sterols in plant cell bi-ology.

However, mutational and transgenic studies have givennew insight into the roles of sterols in plant development.

sterol methyltransferase1

(

smt1

) mutants accumulate cho-lesterol at the expense of sitosterol and cannot be rescuedby BRs (Diener et al., 2000). Transgenic tobacco plants ex-pressing a

35S

::

SMT2

gene accumulate sitosterol at the ex-pense of campesterol, causing reduced stature and growth,and can be rescued by BRs (Schaeffer et al., 2001). Cosup-pression of

SMT2

causes low sitosterol and high campes-terol, accompanied by reduced growth and low fertility,which is not modified by BRs. The reduced growth is linkedwith reduced cell division rather than with the reduced cellelongation that many BR mutants exhibit (Schaeffer et al.,

1

These authors contributed equally to this work.

2

To whom correspondence should be addressed. E-mail keith.lindsey@durham.ac.uk; fax 44-191-374-2417.Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.001248.

1018 The Plant Cell

2001). The identification of sterol binding (SteroidogenicAcute Regulatory Transfer Protein [StART]) domains (Kallenet al., 1998; Ponting and Aravind, 1999) in a number of pat-terning proteins, and in particular some homeodomain pro-teins such as PHABULOSA (PHB/ATHB14), PHAVOLUTA(PHV/ATHB9), GLABRA2 (GL2/ATHB10), REVOLUTA (REV),and ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1),suggests that sterols may modulate regulatory protein func-tion (McConnell et al., 2001). At present, however, a directrole for this domain in these genes remains to be proven.

Two groups have published data on a mutant in the earlypart of the sterol biosynthetic pathway, which is not part ofthe BR pathway and which is not rescued by the exogenousapplication of BRs (Jang et al., 2000; Schrick et al., 2000).This is the

fackel

(

fk

) mutant of Arabidopsis, which encodesa defective sterol C14 reductase, resulting in a pleiotropicphenotype characterized by defective cell shape and divi-sion control in embryogenesis. Schrick et al. (2000) havepostulated that there is a novel, as yet unknown, sterol sig-naling molecule that is not produced in these mutants thatcan explain the phenotype.

In performing a mutational screen to identify genes re-quired for embryo and seedling development in Arabidop-sis, we previously identified a class of seedling-lethal dwarfsthat we termed the

hydra

(

hyd

) mutants (Topping et al.,1997). Recessive mutations at two loci, designated

hyd1

and

hyd2

, resulted in pleiotropic phenotypes, including de-fective cell shape, multiple cotyledons, and short roots andhypocotyls. Because of the phenotypic similarities of thetwo mutants, we predicted that they are defective in thesame or similar pathways required for normal embryo andseedling development.

To gain more insight into the molecular basis of

HYD

genefunction, we aimed to clone the

hyd1

and

hyd2

genes, andin this article, we report the identity of the genes. We showthat the

hyd1

mutant is defective in the plant ortholog ofyeast

erg2

and mammalian EBP,

�

8-

�

7 sterol isomerase.We also show that

hyd2

is allelic to

fk

. Using these mutants,we provide evidence for a role for

�

8-

�

7 sterol isomeraseactivity in eukaryotic morphogenesis and show that sterolsare required for correct auxin and ethylene signaling in Ara-bidopsis, defects that may account for many of the develop-mental abnormalities seen in the mutants.

RESULTS

hyd1

and

hyd2

Are Sterol Biosynthetic Mutants

In a screen of T-DNA and ethyl methanesulfonate mutantsof Arabidopsis, we identified a class of recessive seedling-lethal mutants defined by two loci,

hyd1

and

hyd2

(Toppinget al., 1997). We showed that

hyd1

mutants have a pleiotro-pic phenotype. They are unable to regulate cell size and

shape, and they fail to undergo correct morphogenesis dur-ing embryonic and postembryonic development (Figure 1).Although apical-basal pattern elements are present (shoot,hypocotyl, and root), the radial pattern is defective, with su-pernumerary cell layers and aberrant vascular patterning.Multiple leaf-like cotyledons are produced (Figures 1Ato 1C).

The

hyd2

mutant seedling phenotype is very similar tothat of

hyd1

. In allelism tests, we found that

hyd2

is allelic tothe

fk

mutant, which is defective in the gene encoding thesterol biosynthetic enzyme C-14 sterol reductase (Jang etal., 2000; Schrick et al., 2000). These authors describe theembryonic phenotype, which is essentially identical to thatof

hyd1

(Topping et al., 1997). Using reverse transcriptase–mediated polymerase chain reaction, it was found that the

hyd2

allele of

fk

contained no detectable

FK

transcript andprobably is a null allele caused by a T-DNA insertion that co-segregates with the mutation. Like

hyd1

,

hyd2

/

fk

is dwarfed,with multiple cotyledons, and is seedling lethal. It exhibitsseverely abnormal vascular patterning, as revealed micro-scopically and with the aid of the vascular tissue–specific

�

-glucuronidase (GUS) markers VT-1 (a promoter trapmarker) (Topping et al., 1997) and

PIN1

::

GUS

(which is a fu-sion of the

PIN1

gene promoter and the GUS reporter gene)(Figures 1E and 1F) (L. Gälweiler and K. Palme, unpublisheddata). Commonly, there are a duplicated vascular systemand discontinuities in vascular strands (Figure 1F).

A distinction between the

hyd1

and

hyd2

/

fk

mutants is themore severe root-defective phenotype of

hyd2

/

fk

. Althoughthe

hyd1

root is short and may cease cell division within 2weeks after germination, roots may continue to grow veryslowly until the seedling dies, at

�

30 to 40 days after germi-nation (Topping et al., 1997). In contrast, the

hyd2

/

fk

root in-variably stops growth completely at

�

10 to 14 days aftergermination. Microscopic examination showed a loss ofcorrect cell patterning of the meristem and root cap, with aloss of Lugol staining of the columella, indicating a loss ofthat tissue (Figures 2A to 2D).

Root cell file patterning also was abnormal. The alternat-ing expression of the cell file marker

GL2

::green fluorescentprotein (

GFP

), expressed in atrichoblast (non-hair-forming)files in the wild type (Masucci et al., 1996) (Figure 2E), wasseen in all root epidermal cell files in a

hyd2

/

fk

background(Figure 2F). Interestingly, all cell files produced root hairs,and up to five root hairs can form from individual tricho-blasts (Figure 2G). This finding indicates a defect in themechanisms controlling

GL2

gene expression, and becausethe

GL2

gene is proposed to act as a negative regulator ofhair cell formation (Masucci et al., 1996), it suggests thatGL2 protein activity is either suppressed or bypassed in themutants.

To investigate further the meristematic activity of the

hyd1

and

hyd2

/

fk

seedling roots, heterozygotes were crossedwith Arabidopsis transgenic for a

cyclinB

::

GUS

gene fusioncontaining a cyclin destruction box, which marks individualmitotic cells (Hauser and Bauer, 2000). Although the

hyd1

Sterols and Signaling in Arabidopsis 1019

mutant root showed no obvious change in the frequency ofGUS-positive cells during the 18 days after germination,

hyd2

/

fk

mutants showed a reduced frequency of GUS-posi-tive cells between days 10 and 14 after germination, corre-sponding to the observed disorganization at the root tip atthis time (Figures 2H to 2L). This finding indicates a loss ofmitotic activity of the

hyd2

root meristem during develop-ment.

Two independent

hyd1

alleles,

hyd1-1

and

hyd1-2

, wereidentified previously (Topping et al., 1997). We cloned the

hyd1-2

allele, which was T-DNA tagged (Errampalli et al.,1991). A genomic fragment of 2.6 kb flanking the T-DNA in

hyd1-2

was cloned by plasmid rescue (Behringer and Medford,1992) and found to be 100% homologous with the 5

�

regionof an expressed sequence tag that was identified as a cDNAencoded by the Arabidopsis

�

8-

�

7 sterol isomerase gene(Grebenok et al., 1998). The gene is on chromosome 1. Theenzyme

�

8-

�

7 sterol isomerase is located one step down-stream of the C-14 sterol reductase in the sterol biosyn-

thetic pathway. The predicted protein has high homologywith the mammalian EBP, which is localized to the endo-plasmic reticulum and the nuclear membrane (Dussossoy etal., 1999) and has been demonstrated to have

�

8-

�

7 sterolisomerase activity (Silve et al., 1996a).

A full-length expressed sequence tag clone was obtained,and a genomic clone encompassing the gene was isolatedfrom an Arabidopsis Landsberg genomic library (Voytas etal., 1990). The T-DNA was found to have inserted into thefirst intron of the gene, which most likely resulted in a nullmutation. This finding is supported by the observed lack ofdetectable

HYD1

transcript in the

hyd1-2

mutant, as deter-mined by reverse transcriptase–mediated polymerase chainreaction (data not shown). Sequencing of a second mutantallele (

hyd1-1

) revealed the presence of a point mutationthat causes a Gly-to-Asp substitution at amino acid 66,which falls within a predicted membrane-spanning domain.

To determine whether a wild-type allele of the sterolisomerase gene complements the

hyd1

mutation,

hyd1-2

Figure 1. Morphology and Vascular Patterning of hyd Seedlings.

(A) hyd1 seedling 3 days after germination. Magnification �20.(B) hyd1 seedling 7 days after germination. Magnification �12.(C) hyd2 seedling 5 days after germination. Magnification �15.(D) Cleared wild-type leaf showing PIN1::GUS expression in vascular tissues. Magnification �8.(E) hyd2 leaf showing PIN1::GUS expression in vascular tissues. Magnification �30.(F) hyd2 seedling 3 days after germination showing VT-1 promoter trap expression in vascular tissues. Magnification �15.

1020 The Plant Cell

seedlings, which are heterozygous for the T-DNA insertionalmutation carrying the NPT-II selectable marker, were trans-formed with a wild-type allele of the gene under the tran-scriptional control of 2 kb of its own promoter and carrying aselectable HPT gene, which confers hygromycin resistance.One hundred percent of 40 transgenic F2 progeny, whichcontained both the mutation and the wild-type allele (identi-fiable as being resistant to both kanamycin and hygromycin),were rescued phenotypically (Figure 3A). Transformation ofhyd1-2 homozygous mutants with the wild-type allele linked

to a phosphomannose isomerase selectable marker also ledto phenotypic rescue.

This finding demonstrates that the sterol isomerase wasable to complement the mutation. The wild-type gene underthe transcriptional control of the 35S gene promoter of Cau-liflower mosaic virus (CaMV35S) (compared with the HYD1promoter) was able to rescue the hyd1 mutant only partially,presumably because that promoter is not expressed early inembryogenesis when the hyd1 defects are established. An-tisense expression of the sterol isomerase gene under the

Figure 2. Root Growth and Development in hyd Seedlings.

(A) to (D) Lugol-stained roots of wild-type ([A]; 14 days after germination), hyd1 ([B]; 14 days after germination), hyd2 ([C]; 10 days after germi-nation), and hyd2 ([D]; 14 days after germination) to reveal columella. Magnification �200.(E) and (F) Confocal images of wild-type (E) and hyd2 (F) roots expressing GL2::GFP fusion in epidermal cell files (arrows). Note in hyd2 the ex-pression in all cell files and the abnormal cell shape. Magnification �200.(G) Scanning electron micrograph of a hyd2 root showing ectopic root hairs (arrows) and abnormal root hair shape. Bar � 100 �M.(H) to (L) CyclinB::GUS expression in mitotic cells of roots of wild type (H) and hyd2 at 3 days after germination (I), 12 days after germination (J),15 days after germination (K), and 18 days after germination (L). Magnification �100.

Sterols and Signaling in Arabidopsis 1021

transcriptional control of the CaMV35S promoter caused adwarfed phenotype (Figure 3B). Together, these results con-firm that mutation of the �8-�7 sterol isomerase gene is re-sponsible for the hyd1 phenotype.

A 2-kb fragment upstream of the sterol isomerase codingregion was fused to the gusA reporter gene and introducedinto wild-type Arabidopsis for functional analysis. GUS ac-tivity in transgenic plants was detectable in a developmen-tally regulated pattern during embryogenesis. At the globu-lar and heart stages, GUS activity was constitutive in theembryo proper but was not detectable in the suspensor(Figures 3C and 3D). Torpedo-stage embryos showed lowerlevels of GUS activity (Figure 3E), and cotyledon- and ma-ture-stage embryos had virtually no detectable GUS expres-sion. These data are consistent with the mutant analysis thatindicated the requirement for the sterol isomerase geneearly in embryogenesis, and in the embryo proper, but not inthe suspensor (Topping et al., 1997).

It has been reported previously that fk mutants have ab-normal sterol profiles (Jang et al., 2000; Schrick et al., 2000).We compared the concentrations of the three major sterolsin hyd1 and hyd2/fk seedlings (Table 1). Both hyd1 andhyd2/fk have much reduced levels of sitosterol and campes-terol and increased levels of stigmasterol, similar to other fkalleles. hyd1-1 and hyd1-2 mutants showed no significantdifferences. Sitosterol and campesterol are the most abun-dant sterols in Arabidopsis, each being essential in orderingacyl chains in the membrane lipid bilayer and in regulatingmembrane permeability; stigmasterol cannot functionally re-place either sterol (Schuler et al., 1991; Hartmann, 1998).These results demonstrate the requirement for a functional�8-�7 sterol isomerase activity for sterol composition in Ar-abidopsis and indicate that mutations in sterol isomerase (inhyd1) and in C14 reductase (in hyd2/fk), respectively, resultin similar gross changes in sterol profiles.

To determine whether the exogenous application of sterolscan rescue the mutants, seedlings were grown in the pres-ence of 1, 5, 10, 50, and 100 �M sitosterol, campesterol, orstigmasterol. In no case was any evidence of phenotypic res-cue seen. We showed previously that the hyd mutants are notrescued by BR application (Topping et al., 1997).

hyd1 and hyd2/fk Are Defective in Ethylene and Auxin Signaling Pathways

The pleiotropic phenotypes of hyd1 and hyd2/fk suggestthat correct sterol profiles are required for diverse plant sig-naling pathways to function correctly. For example, the roothair phenotype of hyd2/fk suggests defective ethylene sig-naling, because ethylene is a positive regulator of hair for-mation (Tanimoto et al., 1995), and defective vascularpatterning suggests defective auxin transport or signaling(Mattsson et al., 1999). It also is possible that any failure ofthe hyd/fk mutants to regulate auxin or ethylene signaling,and to respond to exogenous BRs, may be attributable to

defective membrane-dependent processes, such as recep-tor activity, or to defects in permeability leading to the incor-rect tissue distribution of signaling molecules. To determinewhether specific known signaling pathways are altered inthe mutants, we focused on cellular processes in whichauxin and ethylene play important roles.

Ethylene Signaling

Both hyd1 and hyd2/fk roots have root hairs closer to theroot tip than is seen in the wild type, with very abnormal pat-terning evident in hyd2/fk (Figure 2). To investigate whetherethylene signaling is abnormal in the mutant roots, hyd1 andhyd2 mutants were crossed with the ethylene-resistant mu-tant etr1-1 (Figure 4). The double mutants showed a re-stored wild-type pattern of root hair formation (Figures 4Band 4D) and meristem patterning and activity (Figures 4Eand 4F) with sustained growth. Similarly, when hyd1 andhyd2 mutants were grown in the presence of 10 �M silverions, which inhibit ethylene action by binding to the recep-tor (Beyer, 1976), a similar rescue of root structure andgrowth was observed. These results indicate either thathyd2/fk mutants accumulate relatively high levels of ethyl-ene or that the ethylene receptor ETR1 is altered constitu-tively in activity to promote ethylene signaling and that thisis responsible for the observed root hair and defectivemeristem phenotypes.

To determine whether the hyd1 and/or hyd2/fk mutantsproduce abnormal levels of ethylene, seedlings were grownin vitro for 7 days after germination, and evolved ethylenelevels were assayed. In two independent sets of experi-ments, the hyd1 mutants (which are in a C24 background)produced ethylene at mean rates of 3.8 and 21.5 nL·g1

fresh weight·hr1, respectively, compared with mean ratesof 11.9 and 10.8 nL·g1 fresh weight·hr1 for the C24 wildtype. The hyd2 mutant (Wassilewskija background) pro-duced ethylene at mean rates of 15.5 and 6.3 nL·g1 freshweight·hr1, compared with 16.7 and 12.0 nL·g1 freshweight·hr1 for the wild type. These findings indicate thatthe mutants produce variable levels of ethylene but do notproduce dramatically different mean levels of ethylene com-pared with the wild type.

Treatment of mutant seedlings with the ethylene biosyn-thesis inhibitor aminoethoxyvinylglycine (0.5 �M) did notrescue the root or shoot phenotypes in hyd2/fk, consistentwith evidence from the ethylene assays that abnormally highlevels of ethylene biosynthesis are not responsible for theobserved phenotypes; in contrast, hyd1 seedlings showedsome phenotypic rescue of root growth by aminoethoxyvi-nylglycine (data not shown). These data suggest that thehyd2/fk mutants are defective in ethylene signal transduc-tion rather than ethylene biosynthesis, although we cannotexclude the possibility that the mutants produce locally highethylene levels in specific tissues that are not detectable inthe assays.

1022 The Plant Cell

Auxin Signaling

To investigate possible defects in auxin signaling, we ana-lyzed the expression of GUS fusion genes driven by genepromoters previously demonstrated to be responsive to auxin.DR5::GUS and ACS1::GUS, each of which marks auxin-responsive cells in the shoot and hypocotyl, were introducedinto both the hyd1 and the hyd2/fk mutant backgrounds bycrossing. In the wild-type seedling, ACS1::GUS was ex-pressed in developing leaves and in the endodermal layerfrom the hypocotyl to the root expansion zone (Figures 5Aand 5B) and at the site of lateral root initiation (Rodrigues-Pousada et al., 1999). ACS1::GUS was upregulated by auxinin the shoot and upregulated by ethylene and downregulatedby auxin in the root. Expression did not extend into the mer-istem and the root tip. DR5::GUS expression in the shoot wasseen in the hydathodes and developing vasculature only andshowed a “maximum” in the root meristem region in the col-umella initials (Sabatini et al., 1999).

GUS activity in homozygous hyd mutants containing thegene fusions was localized by histochemistry up to 20 days

after germination. Expression was abnormal in a develop-mentally regulated way, with the most dramatic changesseen in hyd2/fk. In hyd2, ACS1::GUS expression was normalin the root until day 6 after germination, when strong GUSactivity was seen throughout the entire root, in all radial lay-ers and extending into the meristem and the root tip (Figure5D). By day 9, this ectopic expression was downregulated,and it was normal for the rest of development (Figure 5E). Inhyd1, ACS1::GUS was expressed in a pattern similar to thatseen in the wild type, although there was more expression inthe shoot. Similarly, in hyd1, the expression of DR5::GUS wasnormal in the root throughout development. hyd2 showednormal expression until days 12 to 14, but after this stage, theexpression was reduced and shifted distally to the meristem;by day 18, there was no expression in the primary root tip(Figure 5). These observations are consistent with alteredauxin distribution, altered distribution of auxin-perceptivecells, and/or altered ethylene signaling in the mutants.

To determine whether the hyd mutants exhibit altered re-sponses to auxin, mutant and wild-type seedlings weregrown in the presence of exogenous 2,4-D (100 nM and 1

Figure 3. HYD1 Gene Function.

(A) Phenotype of a transgenic hyd1 mutant seedling expressing a wild-type allele of the HYD1 sterol isomerase gene under the transcriptionalcontrol of its own promoter. Magnification �4.(B) Wild-type seedling (left) and wild-type seedling transformed with an antisense HYD1 gene under the transcriptional control of the CaMV35Spromoter (right). Magnification �4.(C) to (E) Embryonic expression of the HYD1::GUS promoter fusion in globular-stage (arrow) ([C]; magnification �100), heart-stage ([D]; magni-fication �200) and torpedo-stage ([E]; magnification �200) embryos.

Sterols and Signaling in Arabidopsis 1023

�M) and naphthylacetic acid (1 �M). It was found that bothhyd1 and hyd2/fk mutants underwent a callusing responseat these concentrations of both auxins within 3 to 5 days(Figures 6B and 6C), whereas the wild-type seedlings pro-duced more lateral roots at these concentrations (Figure 6A)but showed evidence of callusing only at 10- to 50-foldhigher auxin concentrations. In hyd1 and hyd2/fk mutantscontaining DR5::GUS and grown similarly in the presence ofauxins, the entire plant/callus stained very strongly for GUS(Figure 6H). In contrast, auxin-treated wild-type seedlingsshowed no dramatic upregulation of GUS activity, exceptspecifically in emerging lateral roots at the higher 2,4-D con-centration (Figure 6E). The observed callusing response andenhanced DR5::GUS activity of the mutants suggest thatthe mutants either are more sensitive to auxins or take upmore exogenous auxins than the wild type.

To determine whether the observed response of the hyd/fk mutants to auxin might be caused by increased cell per-meability, mutant and wild-type seedlings containing DR5::GUS were grown in the presence of 2,4-D in the presenceand absence of the auxin transport inhibitor 1-naphthoxy-acetic acid (1-NOA). 2,4-D uptake requires an influx carrier(Delbarre et al., 1996), and 1-NOA inhibits intracellular up-take of 2,4-D, most likely by inhibiting AUX1 function, andhas been found to inhibit cellular responses to 2,4-D (Parryet al., 2001) (Figure 6D). Seedlings were grown for 3 days af-ter germination in the presence of 10 �M 1-NOA and thentransferred to 10 �M 1-NOA plus 1 �M 2,4-D for another 3days. In both the presence and absence of 1-NOA, the hyd1and hyd2/fk mutants exhibited a callusing response (Figures6G to 6I), demonstrating sensitivity to the hormone and alsoan upregulation of GUS activity that was similar to the up-regulation when grown in the presence of 2,4-D alone.

On the other hand, wild-type seedlings showed reducedDR5::GUS activity when grown on 1-NOA plus 2,4-D (Figure6F) compared with 2,4-D alone (Figure 6E). This finding sug-gests either that 2,4-D can bypass the 1-NOA–sensitiveauxin influx carrier in the hyd/fk mutants, but not in the wildtype, or that a low level of inward leakage of 2,4-D throughthe plasma membrane is sufficient to trigger auxin re-sponses in the mutants but not in wild-type seedlings.These data suggest that some aspects of the hyd mutants,such as altered vascular patterning or defective root devel-opment, might be the result of altered (e.g., ectopic) auxindistribution or signaling.

To investigate whether we could rescue the mutants byblocking auxin action, we performed double mutant analysisbetween hyd1 and hyd2/fk and the auxin-resistant mutantsaxr1-12 and axr3-1, which are defective in the correct re-sponse to auxins (Lincoln et al., 1990; Rouse et al., 1998).Both axr1-12 and axr3-1 have a reduced frequency of roothairs. All double mutants had longer roots and much re-duced root hair production compared with the hyd1 andhyd2/fk single mutants (Figure 6J). The hyd1 axr1-12 andhyd2 axr1-12 double mutants showed partial rescue of leafdevelopment, with longer petioles, less disrupted vascular

patterning, and a smoother leaf surface (Figure 6K), andwere relatively long-lived. There was no obvious effect ofthe axr3-1 mutation on the development of the hyd1 andhyd2 mutant shoots. These results indicate that (1) introduc-ing an auxin-resistant axr1-12 background into the hyd mu-tants ameliorates the abnormal leaf phenotype, and (2) bothaxr1-12– and axr3-1–dependent auxin resistance can re-duce the root defects in the hyd mutants.

hyd/fk Mutants Exhibit Correct Trafficking ofPIN Proteins

It is possible that the observed defective sterol compositionin the hyd1 and hyd2/fk mutants could interfere with correctmembrane trafficking to translocate regulatory (e.g., recep-tor or transport) proteins to the plasma membrane or othercellular compartments. For example, the PIN proteins arepredicted components of the auxin efflux carrier system andare localized precisely to mediate polar auxin transport. As amodel to study vesicle trafficking, we determined whetherthe mutants are defective in the trafficking of PIN1, PIN2,and PIN3 by immunolocalization. In the wild-type Arabidop-sis root, PIN 1 localized to the basal membranes of vascularcells (Gälweiler et al., 1998) (Figure 7A), whereas PIN2 local-ized to the apical membranes in the root cortex and epider-mis (Muller et al., 1998) (Figure 7D). PIN3 was localized tothe first tier of columella cells distal to the columella initialsat all faces of the plasma membrane (J. Friml and K. Palme,unpublished data) (Figure 7G).

It was observed that PIN1 and PIN2 proteins were local-ized normally in hyd1 and hyd2/fk roots (Figures 7A to 7F).Similarly, PIN3 localization was identical to that in the wildtype (Figure 7G) until approximately day 9 after germination(data not shown). However, between days 9 and 12, PIN3localization shifted to the columella initials, and by day 14after germination, it disappeared in parallel with the disorga-nization of the root meristem and columella (Figure 7H).PIN3 localization was rescued in seedlings treated with sil-ver or in hyd2 etr1 double mutants, as demonstrated by the

Table 1. Mean Sterol Concentrations in the Wild Type and hyd1 and hyd2/fk Mutants

SterolWild Type(�g/g fresh weight)

hyd1(% of wild type)

hyd2/fk(% of wild type)

Campesterol 4.20 0.02 12 0Sitosterol 19.51 0.40 2 4Stigmasterol 1.62 0.00 182 322

Pooled seedlings at day 10 after germination were analyzed for thethree major sterols, and the means of triplicate assays SEM arepresented.

1024 The Plant Cell

fact that the root meristem and columella patterning wasrescued (Figure 7I).

DISCUSSION

Sterols Are Required for Correct Plant Morphogenesis

Sterols are known to contribute to two biosynthetic path-ways in plants: the BRs and the bulk sterols (Hartmann,1998; Clouse, 2000). A number of mutants in plant sterolbiosynthetic genes have been identified, most of which havebeen found to encode enzymes required for the synthesis ofBRs. Such mutants typically are dwarfed and can be res-cued phenotypically by exogenous BR application (Szekereset al., 1996; Choe et al., 1999a, 1999b). However, recentstudies identified a sterol mutant, fk, that encodes a defec-tive sterol C14 reductase and is embryonic defective (Janget al., 2000; Schrick et al., 2000). Interestingly, fk mutantsare deficient in BRs but cannot be rescued by their exoge-nous application. Seedlings homozygous for mutant allelesof this gene are very similar phenotypically to the previouslydescribed hyd1 and hyd2 mutants, which also cannot berescued by BR application (Topping et al., 1997). Here, weshow that the hyd2 mutant is allelic to fk.

To further strengthen the view that sterols are required forplant development, we demonstrate here that the HYD1gene encodes the sterol biosynthetic enzyme �8-�7 sterolisomerase, which is found one step downstream of the C14reductase (Grebenok et al., 1998). This proximity of the en-zymes in the pathway presumably accounts for the similari-ties of sterol profiles in the hyd1 and hyd2/fk mutants (Table1) and the similar phenotypes. The subtle differences in ste-rols may account for the observed developmental differ-ences between the mutants. HYD1 occurs upstream ofSMT2, cosuppression of which leads to high campesteroland low sitosterol contents as well as reduced plant growthand fertility, which also cannot be rescued by BR applica-tion (Schaeffer et al., 2001).

The HYD1 sterol isomerase has been cloned previouslyand demonstrated to have sterol isomerase enzyme activity(Grebenok et al., 1998). No corresponding developmentalmutant phenotypes have been identified elsewhere in yeast,plants, or animals. It exhibits regions of strong homologywith mammalian EBP, a membrane-bound �1 factor recep-tor with orthologs identified previously in mouse (Silve et al.,1996a), human (Hanner et al., 1995; Jbilo et al., 1997), andguinea pig (Hanner et al., 1995). EBP is resident in the endo-plasmic reticulum and the nuclear envelope (Jbilo et al.,1997; Dussossoy et al., 1999).

Both the Arabidopsis and mammalian sterol isomerasegenes complement the yeast erg2 mutant defective in thisenzyme (Silve et al., 1996a; Grebenok et al., 1998), althoughEBP has no significant structural homology with yeast sterolisomerase and was thought previously to be unique to

Figure 4. hyd Root Phenotype Is Rescued Partially by InhibitingEthylene Action.

(A) Wild-type (left), etr1-1 (second from left), hyd2 etr1-1 (third fromleft), and hyd2 (right) seedlings showing increased root length in thedouble mutant compared with the hyd2 mutant.(B) to (D) Roots of hyd2 (B), the wild type (C), and hyd2 etr1-1 (D)showing root hairs. Magnification �10.(E) hyd2 root 14 days after germination stained with Lugol to revealthat the columella is lacking. Magnification �200.(F) hyd2 etr101 root 14 days after germination stained with Lugol.Magnification �200.

Sterols and Signaling in Arabidopsis 1025

mammals. However, EBP is now believed to be the mam-malian �8-�7 sterol isomerase. The HYD1 protein, in com-mon with EBP, has four predicted membrane-spanningdomains and a consensus endoplasmic reticulum retrievaldomain at the C terminus, K(X)KXX. There are several otherconserved sequence domains, with one being very highlyconserved (60% identical and 91% similar over amino acids94 to 115). The activity of the HYD1 promoter in transgenicembryos is consistent with the embryonic phenotype of thehyd1 mutant.

EBP has high affinity for emopamil, a phenylalkylamineCa2� antagonist (Hanner et al., 1995), and for trifluopera-zine, an inhibitor of calcium signaling pathways in animalsand plants. It has been proposed that the effect on calciumsignaling is mediated via alterations to membrane sterolcomposition that affect correct calcium transport acrossmembranes or, possibly, calmodulin action at the mem-brane (Silve et al., 1996a). The hypersensitivity of the smt1mutant of Arabidopsis to calcium, which has reduced levelsof sitosterol and other C24 sterols (Diener et al., 2000), cor-relates with defective root growth and may similarly reflectaltered membrane permeability to, or perception of, this ion.

Pharmacological inhibition of the yeast C-14 sterol reduc-tase ERG24 (Marcireau et al., 1990) and mammalian �8-�7sterol isomerase in recombinant yeast and in mammaliancells (Silve et al., 1996b) leads to cell cycle arrest. In con-trast to the essential yeast ERG24, ERG2 (yeast sterolisomerase) has been found to be functionally redundant(Ashman et al., 1991; Lees et al., 1995). Therefore, the hyd1mutation provides the first direct evidence of a requirementfor �8-�7 sterol isomerase in eukaryotic morphogenesis.

Molecular Basis for hyd/fk Defects

A key question is how defects in sterol biosynthesis arelinked to the pleiotropic developmental defects seen in thehyd1 and hyd2/fk mutants. A number of possibilities can beconsidered. It is possible that BRs, or sterols with hormone-like activities missing in the mutants, are required during de-velopment. For example, specific sterols that are reduced inthe mutants may have signaling roles directly or indirectly,such as through interaction with sterol binding domains inproteins such as PHABULOSA, which is known to play an

Figure 5. hyd Mutants Show Altered Gene Expression.

(A) to (E) ACS1::GUS expression in the wild type 3 days after germination ([A] and [B]), in hyd2 3 days after germination (C), in hyd2 6 days aftergermination (D), and in hyd2 9 days after germination (E). Magnification �5 (A) and �200 (B) to (E).(F) to (I) DR5::GUS expression in the wild type 3 days after germination (F), in hyd2 3 days after germination (G), in hyd2 6 days after germination(H), and in hyd2 9 days after germination (I). Magnification �100.

1026 The Plant Cell

Figure 6. hyd Mutants Show Altered Responses to Auxin.

(A) to (C) Response of wild-type (A), hyd1 (B), and hyd2 (C) seedlings on 1 �M naphthylacetic acid.(D) Wild-type seedlings transgenic for DR5::GUS treated with 1 �M 2,4-D (left) and 1 �M 2,4-D plus 10 �M 1-NOA (right).(E) and (F) Roots of wild-type seedlings transgenic for DR5::GUS treated with 1 �M 2,4-D (E) and 1 �M 2,4-D plus 10 �M 1-NOA (F).(G) to (I) hyd2 (G) and hyd2::DR5-GUS transgenic plants treated with 1 �M 2,4-D (H) and 1 �M 2,4-D plus 10 �M 1-NOA ([I]).(J) hyd2 axr3 root 18 days after germination showing lack of root hairs and less defective cellular organization.(K) Cleared hyd1 axr1-12 leaves showing rescue of petiole length, morphology, and vascular pattern (cf. with Figure 1E).Magnification �4 (A), �10 ([B] and [C]), �6 (D), �16 ([E] and [F]), �15 ([G] and [I]), �18 (H), �40 (J), and �10 (K).

Sterols and Signaling in Arabidopsis 1027

important role in Arabidopsis development (McConnell etal., 2001).

Furthermore, fk mutants have been shown to accumulatelow levels of BRs, which could contribute to the dwarfed fkphenotype (Jang et al., 2000; Schrick et al., 2000). However,neither hyd1 nor hyd2/fk mutants can be rescued by the ex-ogenous application of BRs, although mutants defective inenzymes closer to the branch of the pathway that is com-mitted specifically to BR biosynthesis can. This suggeststhat the hyd/fk mutants, which are defective in bulk sterolbiosynthesis, with low campesterol as well as low sitosteroland high stigmasterol, are defective in the perception or sig-nal transduction of exogenous BRs, a process mediated bythe membrane-bound receptor kinase BRI1 (Li and Chory,1997).

Additionally, it was not possible to rescue hyd/fk mutantsby the exogenous application of bulk sterols. Although thelimited uptake or transport of sterols could prevent rescue,this may be unlikely because exogenous BRs can rescueBR mutants (Clouse, 2000). It also is possible that the em-bryonic defects would not be corrected by sterol applicationat the seedling stage, although HYD1 is expressed in seed-lings (J. Topping, M. Pullen, X.-L. Zhang, and K. Lindsey,unpublished data), indicating a requirement for sterol bio-synthesis after germination. However, we found no evidenceof any alteration in the development of the mutant seedlingsby such treatments.

Sterols could influence the function of transport vesiclesand thus the trafficking of important signaling or other pro-teins to membrane compartments, and this process couldbe disrupted in sterol mutants. However, analysis of PINprotein localization indicates that their targeting to theplasma membrane is not affected detectably in either thehyd1 or the hyd2/fk mutant. This finding suggests that the de-fective sterol composition of the mutants does not interferewith correct vesicle transport to the plasma membrane;therefore, the observed spatial defects in auxin-mediatedgene expression patterns are not caused by altered PIN lo-calization.

We have shown that both hyd1 and hyd2/fk mutants aredefective in ethylene- and auxin-mediated gene expressionand cell differentiation. Both hyd1 and hyd2/fk mutants cal-lus at lower auxin concentrations than do wild-type seed-lings. The observation that 1-NOA, an inhibitor of auxininflux, can reduce 2,4-D responses in the wild type but notin hyd mutants suggests that either the NOA-sensitive com-ponent of auxin influx, likely AUX1 (Delbarre et al., 1996;Yamamoto and Yamamoto, 1998; Parry et al., 2001), is by-passed or the hyd mutants are altered in auxin sensitivityrather than increased in membrane permeability to auxin.Because 2,4-D can bypass the influx carrier, albeit at low ef-ficiency, it is possible that in NOA-treated mutants, suffi-cient auxin enters the cell to elicit the callusing responsebecause of enhanced auxin perception or signal transduc-tion. This view is supported by the increased sensitivity ofhyd mutants to naphthylacetic acid, which does not require

AUX1-mediated import. The partial rescue of hyd mutantleaf morphology by the axr1 mutation and of the root de-fects by the axr1 and axr3 mutations, both of which lead toan inability to elicit some auxin responses (Leyser, 1998),further supports the view that the mutants exhibit ectopicauxin action.

The root hair phenotype of the mutants also suggests al-tered ethylene and/or auxin signaling. Interestingly, GL2::green fluorescent protein expression in the hyd/fk mutantsshowed ectopic expression in hair-producing cell files (Figure2F). Because GL2 is a transcription factor that is expressednormally in atrichoblasts of the root epidermis and is con-sidered a negative regulator of hair formation (Masucci etal., 1996), it would be expected that its ectopic expressionpattern would result in fewer, rather than more, hair-produc-ing cell files. Because the GL2 protein has a predicted sterolbinding domain (Ponting and Aravind, 1999; McConnell etal., 2001), it is possible that its activity is suppressed by alack of correct sterol ligands in the hyd/fk mutants, resultingin a glabrous root phenotype. However, it is known that thehormones auxin and ethylene act downstream of GL2 toregulate hair formation (Masucci and Schiefelbein, 1996),and it is possible that the observed auxin and ethylene re-sponse defects in the mutants bypass the ectopic GL2 ex-pression to promote the glabrous phenotype. The observedrescue of hyd/fk root cell patterning by the inhibition of auxinand ethylene action supports this view.

The ethylene assays show that the mutants do not detect-ably overproduce ethylene, and the ethylene biosynthesis in-hibitor aminoethoxyvinylglycine fails to rescue the hyd2/fkmutant phenotype, with only partial rescue of hyd1 roots. Weconclude that the mutants either overproduce ethylene at lowlevels, such as in a tissue-specific way not detectable by theassays, or exhibit altered ethylene signaling, perhaps as a re-sult of a conformational change in the ethylene receptor(s),which is rescued by the etr1-1 mutation (which is dominant;Hall et al., 1999), or by silver, which also may modify ETR1conformation or the signal propagation to the kinase domainof the receptor.

In summary, our studies provide strong evidence that de-fects in plant sterol profiles can lead to defective hormonesignaling. The results of the auxin/1-NOA, aminoethoxyvi-nylglycine, and double mutant experiments in particularsupport the view that the hyd/fk mutants are defective inmembrane-bound protein activity (i.e., defective auxin/ethyl-ene receptor function) or membrane permeability to auxin.The lack of response to exogenous BRs similarly suggestsdefective signaling mediated by the membrane-bound BRI1.It has been shown previously that membrane sterol contentcan modulate the activity of membrane-bound proteins,such as the plasma membrane H�-ATPase from maizeroots, a protein required for ion and auxin transport, cell wallacidification, and cell expansion (Grandmougin-Ferjani etal., 1997). Furthermore, sterols can affect ATPase activitythrough the modulation of fatty acyl chains and membranefluidity (Cooke and Burden, 1990). In animal cells, cholesterol

1028 The Plant Cell

can reorganize lipid packing in the bilayer and create do-mains of varying lipid composition, leading to altered en-zyme activity (Carruthers and Melchior, 1986).

One interesting question is why mutants defective rela-tively early in sterol biosynthesis (hyd1, hyd2/fk, and smt1)have severely aberrant embryogenesis, whereas mutantsthat affect enzymes closer to BR biosynthesis (dwf1 anddwf7) (Choe et al., 1999a, 1999b) do not. It is possible thatthe DWF1 and DWF7 genes are not required during embryo-genesis or are redundant during that phase of development,whereas HYD1, HYD2/FK, and SMT1 are functional andnonredundant. It also is possible that, as alluded to above,hyd1, hyd2/fk, and smt1 lack sterol-based signaling mole-cules, or sterols required for membrane-bound signalingprotein function, that are functionally distinct from BRs, andthat these are essential for correct embryogenesis. The roleof sterols in plant cell signaling likely will be a fruitful area offuture investigation.

METHODS

Mutants and Marker Lines

The hyd1 and hyd2 mutants were identified in a screen of transgeniclines as described previously (Topping et al., 1997). Genetic crosseswere performed with a stereomicroscope, using fine watchmaker’sforceps to dissect floral buds, remove immature anthers, and trans-fer mature pollen from the male parent to the stigma. For in vitrogrowth studies, Arabidopsis thaliana seed were vernalized, surface-sterilized, and plated on growth medium (half-strength Murashigeand Skoog [1962] medium [1/2MS10; Sigma], 1% Suc, and 3.25 g/Lphytagel agar [Sigma]) as described (Topping et al., 1997). For hor-mone/inhibitor application experiments, seed were germinatedaseptically on growth medium containing various concentrations ofhormones and assayed according to the particular experiment. Tis-sue localization of �-glucuronidase enzyme activity was performedas described (Topping et al., 1997).

Figure 7. PIN Localization Is Unaffected in hyd Mutants.

(A) to (C) PIN1 localization (arrow) in wild-type (A), hyd1 (B), and hyd2 (C) roots 6 days after germination.(D) to (F) PIN2 localization (arrows) in wild-type (D), hyd1 (E), and hyd2 (F) roots 6 days after germination.(G) to (I) PIN3 localization (arrow) in wild-type (G), hyd2 ([H]; 14 days after germination), and hyd2 etr1-1 ([I]; 14 days after germination) roots 6days after germination.Magnification �200.

Sterols and Signaling in Arabidopsis 1029

Cloning of the HYD1 Gene

The HYD1 gene was identified as being putatively T-DNA tagged bycosegregation of the mutant phenotype and T-DNA from the vector3850:1003 (Errampalli et al., 1991). The T-DNA vector contains aNPT-II gene conferring kanamycin resistance to seedlings, whichwere plated on 1/2MS10 supplemented with 25 mg/L kanamycin sul-fate. Sixty of 60 (100%) hyd1 seedlings were demonstrated to bekanamycin resistant, and 124 of 205 (61%) phenotypically wild-typeseedlings, progeny from a selfed hyd heterozygote, were kanamycinresistant. This finding indicated that the T-DNA was present at a sin-gle genetic locus. DNA gel blot analysis on the progeny of a hyd1-2heterozygote demonstrated that a single copy of the T-DNA waspresent in 25 of 25 of the heterozygotes and 0 of 10 of the segregat-ing wild types. T-DNA flanking sequence was cloned by plasmid res-cue (Behringer and Medford, 1992). Genomic DNA from an individualhyd1-2 heterozygote plant was digested with SalI to allow left borderrescues.

DNA fragments were cloned in DH5� Escherichia coli cells, and aleft border clone, containing �2.6 kb of flanking plant genomic DNA,was identified by colony hybridization. The genomic DNA was ampli-fied by direct polymerase chain reaction using primers located in thepBR322 origin of the replication region and the left border of the T-DNAand cloned directly into the pCRII vector (Invitrogen, Carlsbad, CA).Nucleotide and deduced protein sequences were used to search forhomologies in the National Center for Biotechnology Information nu-cleotide and protein sequence databases using the BLAST networkservice (Altschul et al., 1990). A corresponding �8-�7 sterol isomeraseexpressed sequence tag was obtained from the ABRC (Columbus,OH) and demonstrated to be a full-length cDNA. The position of theT-DNA in the gene was determined by sequence analysis of the plas-mid-rescued T-DNA–genome junctions.

The full-length sterol isomerase cDNA was cloned as a transcrip-tional fusion under the control of either the 35S promoter of Cauli-flower mosaic virus or its own promoter in the T-DNA binary vectorpCIRCE (a gift from M. Bevan, John Innes Centre, Norwich, UK)containing either the NPT-II gene for kanamycin selection or thephosphomannose isomerase gene (Joersbo et al., 1998) for retrans-formation of kanamycin-resistant mutants. Seed transformed withconstructs containing the phosphomannose isomerase gene wereselected on 1/2MS10 plates supplemented with 4 g/L D-Man.

Wild-type Columbia and hyd1 heterozygotes were transformed bythe floral dip method (Clough and Bent, 1998) using Agrobacteriumtumefaciens strain C58C3 (Dale et al., 1989) and also by the in vitroroot transformation method using Agrobacterium strain LBA4404(Clarke et al., 1992). The morphologies of the resulting transformantswere identical in either transformation procedure. hyd1 mutants weretransformed in vitro using a modified version of the cotyledon explantmethod described by Schmidt and Willmitzer (1991). Whole hyd1mutants including the roots were grown on 1/2MS10 for 14 days,chopped into �1-mm2 pieces, and cultured on callus-inducing me-dium plates without a feeder layer.

After preculture, the explants were incubated with Agrobacteriumstrain LBA4404 harboring either the 35S Cauliflower mosaic virus–driven sterol isomerase cDNA or 2 kb of the genomic sterolisomerase promoter region driving the sterol isomerase cDNA. Agro-bacteria were grown in Luria-Bertani medium supplemented with 10mg/L acetosyringone. After Agrobacterium inoculation, the hyd1 ex-plants were transferred to shoot-inducing medium (Schmidt andWillmitzer, 1991) without Man/hygromycin selection. Selection wasomitted to promote regeneration of the transformed tissue; nonin-fected hyd1 explants were used as a control.

For sterol feeding experiments, seedlings were germinated andgrown for 21 days on 1/2MS10 supplemented with 0 to 100 �Mcampesterol, stigmasterol, and sitosterol (Sigma) dissolved in 1%dimethyl formamide. Sterol assays in extracts of hyd mutants andwild-type seedlings (10 days after germination) were performed bygas chromatography in triplicate pooled samples by the commercialLipid Analytical Service at the Scottish Crops Research Institute(Dundee, UK). For ethylene assays, wild-type and mutant seedlingswere germinated and grown for 7 days on 1/2MS10 and then trans-ferred for 5 hr to sealed vials, where air was sampled and ethyleneevolution was assayed, as described (Llop-Tous et al., 2000). Assayswere performed on populations of five seedlings per vial, and be-tween two and four vials were assayed for each mutant/wild-typepair. These experiments were performed in duplicate.

Immunolocalization and Microscopy

Both stained and unstained tissues were fixed for 15 min in a solutionof 0.24 N HCl and 20% methanol at 57 C. This was replaced with a so-lution of 7% NaOH and 60% ethanol and incubated for 15 min at roomtemperature. Samples then were rehydrated for 5 min each in 40, 20,and 10% ethanol and infiltrated for 15 min in 5% ethanol and 25%glycerol. Samples were mounted in 50% glycerol and viewed withZeiss Axioskop (Jena, Germany), Nikon Optiphot (Tokyo, Japan; differ-ential interference contrast optics), or Olympus SZH10 (Tokyo, Japan)microscopes. Images were captured on Ektachrome 160T film (Kodak,Rochester, NY) and processed in Photoshop 4.0 (Adobe Systems,Mountain View, CA).

For immunolocalization of PIN proteins, tissue was fixed in 4%paraformaldehyde/MTSB (50 mM Pipes, 5 mM EGTA, 5 mM MgSO4,pH 6.9 to 7.0) under vacuum for 1 hr and washed with MTSB/TritonX-100 (0.1%) five times and then with water/Triton X-100 (0.1%) fivetimes. Tissue was permeabilized in a 2% Driselase/MTSB solutionfor 30 min at room temperature and then washed with MTSB/TritonX-100 (0.1%) five times. Samples were incubated for 1 hr in 10%DMSO/Nonidet P-40 (0.5%)/MTSB, washed with MTSB/Triton X-100(0.1%) five times, and incubated for 1 hr in 3% BSA/MTSB.

Tissue was incubated with anti-PIN primary antibody in 3% BSA/MTSB for 4 hr, washed five times in MTSB/Triton X-100 (0.1%), andthen incubated with fluorescein isothiocyanate–labeled secondaryantibody in 3% BSA/MTSB for 3 hr. Samples were washed five timeswith MTSB/Triton X-100 (0.1%) and then five times with water. Sam-ples were mounted on slides in a glycerol-based Slowfade antifadeagent (Molecular Probes, Eugene, OR) and viewed on a LeicaDMIRBE TCS 4D confocal microscope (Wetzlar, Germany), with flu-orescein isothiocyanate–specific detection at 530 15 nm and au-tofluorescence-specific detection at 580 15 nm. Images werecaptured and exported to Photoshop 4.0, in which the two imageswere overlaid.

For Lugol staining of roots, tissues were incubated in Lugol solu-tion (Sigma) for 5 to 10 min and mounted in chloral hydrate (Toppingand Lindsey, 1997) or 20% glycerol for microscopic analysis.

ACKNOWLEDGMENTS

We thank Dr. Ken Feldmann for providing prospective hyd alleles, Dr.Jane Murfett for providing DR5::GUS seed, Dr. D. Van Der Straetenfor providing ACS1::GUS seed, Dr. John Schiefelbein for providingGL2::GFP seed, and Dr. Ottoline Leyser for axr1-12 and axr3-1 seed.

1030 The Plant Cell

etr1 and fk seed was obtained from the Nottingham ArabidopsisStock Centre. This work was supported by a Biotechnology and Bio-logical Science Research Council research studentship to M.S., aDurham University studentship to M.P., and Biotechnology and Bio-logical Science Research Council Grant 12/P02330 to J.T.

Received December 13, 2001; accepted February 8, 2002.

REFERENCES

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J.(1990). Basic local alignment search tool. J. Mol. Biol. 215, 403–410.

Ashman, W.H., Barbuch, R.J., Ulbright, C.E., Jarrett, H.W., andBard, M. (1991). Cloning and disruption of the yeast C-8 sterolisomerase. Lipids 26, 628–632.

Behringer, F.J., and Medford, J.I. (1992). A plasmid rescue tech-nique for the recovery of plant DNA disrupted by T-DNA insertion.Plant Mol. Biol. Rep. 10, 190–198.

Beyer, E.M.J. (1976). A potent inhibitor of ethylene action in plants.Plant Physiol. 58, 268–271.

Carayon, P., Bouaboula, M., Loubet, J.F., Bourrie, B., Petitpretre,G., Le Fur, G., and Casellas, P. (1995). The sigma ligand 31747prevents the development of acute graft-versus-host disease inmice by blocking IFN-gamma and GM-CSF mRNA expression.Int. J. Immunopharmacol. 17, 753–761.

Carruthers, A., and Melchior, D.L. (1986). How bilayer lipids affectmembrane-protein activity. Trends Biochem. Sci. 11, 331–335.

Choe, S., Dilkes, B.P., Gregory, B.D., Ross, A.S., Yuan, H., Noguchi,T., Fujioka, S., Takatsuto, S., Tanaka, A., Yoshida, S., Tax, F.E.,and Feldmann, K.A. (1999a). The Arabidopsis dwarf1 mutant isdefective in the conversion of 24-methylenecholesterol tocampesterol in brassinosteroid biosynthesis. Plant Physiol. 119,897–907.

Choe, S., Noguchi, T., Fujioka, S., Takatsuto, S., Tissier, C.P.,Gregory, B.D., Ross, A.S., Tanaka, A., Yoshida, S., Tax, F.E.,and Feldmann, K.A. (1999b). The Arabidopsis dwf7/ste1 mutantis defective in the �7 sterol C-5 desaturation step leading tobrassinosteroid synthesis. Plant Cell 11, 207–221.

Clarke, M.C., Wei, W., and Lindsey, K. (1992). High frequencytransformation of Arabidopsis thaliana by Agrobacterium tumefa-ciens. Plant Mol. Biol. Rep. 10, 178–189.

Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified methodfor Agrobacterium-mediated transformation of Arabidopsisthaliana. Plant J. 16, 735–743.

Clouse, S.D. (2000). Plant development: A role for sterols inembryogenesis. Curr. Biol. 10, R601–R604.

Cooke, D.J., and Burden, R.S. (1990). Lipid modulation of plasma-membrane-bound ATPases. Physiol. Plant. 78, 153–159.

Dale, P.J., Marks, M.S., Brown, M.M., Woolston, C.J., Gunn, H.V.,Mullineaux, P.M., Lewis, D.M., Kemp, J.M., and Chen, D.(1989). Agroinfection of wheat: Inoculation of in vitro grown seed-lings and embryos. Plant Sci. 63, 237–245.

Delbarre, A., Muller, P., Imhoff, V., and Guern, J. (1996). Compari-son of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid, naphthalene-1-acetic acid, and

indole-3-acetic acid in suspension-cultured tobacco cells. Planta198, 532–541.

Derocq, J.M., Bourrie, B., Segui, M., Le Fur, G., and Casellas, P.(1995). In vivo inhibition of endotoxin-induced pro-inflammatorycytokines production by the sigma ligand SR 31747. J. Pharma-col. Exp. Ther. 272, 224–230.

Diener, A.C., Li, H., Zhou, W.-x., Whoriskey, W.J., Nes, W.D., andFink, G.R. (2000). STEROL METHYLTRANSFERASE 1 controlsthe level of cholesterol in plants. Plant Cell 12, 853–870.

Dussossoy, D., Carayon, P., Belugou, S., Feraut, D., Bord, A.,Goubet, C., Roque, C., Vidal, H., Combes, T., Loison, G., andCasellas, P. (1999). Colocalization of sterol isomerase and sigma1

receptor at endoplasmic reticulum and nuclear envelope level.Eur. J. Biochem. 263, 377–385.

Errampalli, D., Patton, D., Castle, L., Mickelson, L., Schnall, J.,Feldmann, K., and Meinke, D. (1991). Embryonic lethals and T-DNAinsertional mutagenesis in Arabidopsis. Plant Cell 3, 149–157.

Gachotte, D., Husselstein, T., Bard, M., Lacroute, F., and Benveniste,P. (1996). Isolation and characterization of an Arabidopsis thalianacDNA encoding a �7-sterol-C-5-desaturase by functional comple-mentation of a defective yeast mutant. Plant J. 9, 391–398.

Gälweiler, L., Guan, C., Muller, A., Wisman, E., Mendgen, K.,Yephremov, A., and Palme, K. (1998). Regulation of polar auxintransport by AtPIN1 in Arabidopsis vascular tissue. Science 282,2226–2230.

Grandmougin-Ferjani, A., Schuler-Muller, I., and Hartmann, M.-A.(1997). Sterol modulation of the plasma membrane H�-ATPaseactivity from corn roots reconstituted into soybean lipids. PlantPhysiol. 113, 163–174.

Grebenok, R.J., Ohnmeiss, T.E., Yamamoto, A., Huntley, E.D.,Galbraith, D.W., and Della Penna, D. (1998). Isolation and char-acterization of an Arabidopsis thaliana C-8,7 sterol isomerase:Functional and structural similarities to mammalian C-8,7 sterolisomerase/emopamil-binding protein. Plant Mol. Biol. 38, 807–815.

Hall, A.E., Chen, Q.H.G., Findell, J.L., Schaller, G.E., andBleecker, A.B. (1999). The relationship between ethylene bindingand dominant insensitivity conferred by mutant forms of the ETR1ethylene receptor. Plant Physiol. 121, 291–299.

Hanner, M., Moebius, F.F., Webers, F., Grabner, M., Striessnig,J., and Glossman, H. (1995). Phenylalkylamine Ca2� antagonistbinding protein. J. Biol. Chem. 270, 7551–7557.

Hartmann, M.-A. (1998). Plant sterols and the membrane environ-ment. Trends Plant Sci. 3, 170–175.

Hauser, M.-T., and Bauer, E. (2000). Histochemical analysis of rootmeristem activity in Arabidopsis thaliana using a cyclin:GUS(�-glucuronidase) marker line. Plant Soil 226, 1–10.

Jang, J.-C., Fujioka, S., Tasaka, M., Seto, H., Takatsuto, S., Ishii,A., Aida, M., Yoshida, S., and Sheen, J. (2000). A critical role ofsterols in embryonic patterning and meristem programmingrevealed by the fackel mutants of Arabidopsis thaliana. GenesDev. 14, 1485–1497.

Jbilo, O., et al. (1997). Purification and characterization of the humanSR 31747A-binding protein: A nuclear membrane protein relatedto yeast sterol isomerase. J. Biol. Chem. 272, 27107–27115.

Joersbo, M., Donaldson, I., Kreiberg, J., Guldager Peterson, S.,Brunstedt, J., and Okkels, F.T. (1998). Analysis of mannoseselection used for transformation of sugar beet. Mol. Breeding 4,111–117.

Sterols and Signaling in Arabidopsis 1031

Kallen, C.B., Billheimer, J.T., Summers, S.A., Stayrook, S.E.,Lewis, M., and Strauss, J.F. (1998). Steroidogenic acute regula-tory protein (StAR) is a sterol transfer protein. J. Biol. Chem. 273,26285–26288.

Lees, N.D., Skaggs, B., Kirsch, D.R., and Bard, M. (1995). Cloningof the late genes in the ergosterol biosynthetic pathway of Sac-charomyces cerevisiae: A review. Lipids 30, 221–226.

Leyser, O. (1998). Auxin signalling: Protein stability as a versatilecontrol target. Curr. Biol. 8, R305–R307.

Li, J., and Chory, J. (1997). A putative leucine-rich repeat receptorkinase involved in brassinosteroid signal transduction. Cell 90,929–938.

Lincoln, C., Britton, J.H., and Estelle, M. (1990). Growth anddevelopment of the axr1 mutants of Arabidopsis. Plant Cell 2,1071–1080.

Llop-Tous, I., Barry, C.S., and Grierson, D. (2000). Regulation ofethylene biosynthesis in response to pollination in tomato flowers.Plant Physiol. 123, 971–978.

Marcireau, C., Guilloton, M., and Karst, F. (1990). In vivo effects offenpropimorph on the yeast Saccharomyces cerevisiae and deter-mination of the molecular basis of the antifungal property. Antimi-crob. Agents Chemother. 34, 989–993.

Masucci, J.D., and Schiefelbein, J.W. (1996). Hormones act down-stream of TTG and GL2 to promote root hair outgrowth duringepidermis development in the Arabidopsis root. Plant Cell 8,1505–1517.

Masucci, J.D., Rerie, W.G., Foreman, D.R., Zhang, M., Galway,M.E., Marks, M.D., and Schiefelbein, J.W. (1996). Thehomeobox gene GLABRA2 is required for position-dependent celldifferentiation in the root epidermis of Arabidopsis thaliana. Devel-opment 122, 1253–1260.

Mattsson, J., Sung, Z.R., and Berleth, T. (1999). Responses ofplant vascular systems to auxin transport inhibition. Development126, 2979–2991.

McConnell, J.R., Emery, J., Eshed, Y., Bao, N., Bowman, J., andBarton, M.K. (2001). Role of PHABULOSA and PHAVOLUTA indetermining radial patterning in shoots. Nature 411, 709–713.

Muller, A., Guan, C.H., Galweiler, L., Tanzler, P., Huijser, P.,Marchant, A., Parry, G., Bennett, M., Wisman, E., and Palme,K. (1998). AtPIN2 defines a locus of Arabidopsis for root gravitro-pism control. EMBO J. 17, 6903–6911.

Murashige, T., and Skoog, F. (1962). A revised medium for rapidgrowth and bioassays with tobacco tissue culture. Physiol. Plant.15, 473–497.

Parry, G., Delbarre, A., Marchant, A., Swarup, R., Perrot-Rechenmann, C., and Bennett, M. (2001). Physiological charac-terization of a novel class of auxin influx carrier inhibitors. Plant J.25, 399–406.

Ponting, C.P., and Aravind, L. (1999). START: A lipid bindingdomain in StAR, HD-ZIP and signalling proteins. Trends Biochem.Sci. 24, 130–132.

Rodrigues-Pousada, R., Van Caeneghem, W., Chauvaux, N., VanOnekelen, H., Van Montagu, M., and Van Der Straeten, D. (1999).Hormonal cross-talk regulates the Arabidopsis thaliana 1-amino-cyclopropane-1-carboxylate synthase gene 1 in a developmentaland tissue-dependent manner. Physiol. Plant. 105, 312–320.

Rouse, D., Mackay, P., Stirnberg, P., Estelle, M., and Leyser, O.(1998). Changes in auxin response from mutations in an AUX/IAAgene. Science 279, 1371–1373.

Sabatini, S., Beis, D., Wolkenfelt, H., Murfett, J., Guilfoyle, T.,Malamy, J., Benfey, P., Leyser, O., Bechtold, N., Weisbeek, P.,and Scheres, B. (1999). An auxin-dependent distal organizer ofpattern and polarity in the Arabidopsis root. Cell 99, 463–472.

Schaeffer, A., Bronner, R., Benveniste, P., and Schaller, H.(2001). The ratio of campesterol to sitosterol that modulatesgrowth in Arabidopsis is controlled by STEROL METHYLTRANS-FERASE 2;1. Plant J. 25, 605–615.

Schmidt, R., and Willmitzer, L. (1991). Arabidopsis regenerationand transformation (leaf and cotyledon explant system). In PlantTissue Culture Manual: Fundamentals and Applications, Vol. A6,K. Lindsey, ed (Dordrecht, The Netherlands: Kluwer AcademicPublishers), pp. 1–17.

Schrick, K., Mayer, U., Horrichs, A., Kuhnt, C., Bellini, C., Dangl,J., Schmidt, J., and Jürgens, G. (2000). FACKEL is a sterol C-14reductase required for organized cell expansion in Arabidopsisembryogenesis. Genes Dev. 14, 1471–1484.

Schuler, I., Milon, A., Nakatini, Y., Ourisson, G., Albrecht, A.M.,Beneniste, P., and Hartmann, M.-A. (1991). Differential effects ofplant sterols on water permeability and on acyl chain ordering ofsoybean phosphatidylcholine bilayers. Proc. Natl. Acad. Sci. USA88, 6926–6930.

Silve, S., Dupuy, P.H., Labit-Lebouteiller, C., Kaghad, M., Chalon,P., Rahier, A., Taton, M., Lupker, J., Shire, D., and Loison, G.(1996a). Emopamil-binding protein, a mammalian protein thatbinds a series of structurally diverse neuroprotective agents,exhibits a �8-�7 sterol isomerase activity in yeast. J. Biol. Chem.271, 22434–22440.

Silve, S., et al. (1996b). The immunosuppressant SR 31747 blockscell proliferation by inhibiting a steroid isomerase in Saccharomy-ces cerevisiae. Mol. Cell. Biol. 16, 2719–2727.

Szekeres, M., Nemeth, K., Koncz-Kalman, Z., Mathur, J.,Kauschmann, A., Altmann, T., Redei, G.P., Nagy, F., Schell, J.,and Koncz, C. (1996). Brassinosteroids rescue the deficiency ofCYp90, a cytochrome P450, controlling cell elongation and de-eti-olation in Arabidopsis. Cell 85, 171–182.

Tanimoto, M., Roberts, K., and Dolan, L. (1995). Ethylene is a pos-itive regulator of root hair development in Arabidopsis thaliana.Plant J. 8, 943–948.

Topping, J.F., and Lindsey, K. (1997). Promoter trap markers differ-entiate structural and positional components of polar develop-ment in Arabidopsis. Plant Cell 9, 1713–1725.

Topping, J.F., May, V.J., Muskett, P.R., and Lindsey, K. (1997).Mutations in the HYDRA1 gene of Arabidopsis perturb cell shapeand disrupt embryonic and seedling morphogenesis. Develop-ment 124, 4415–4424.

Voytas, D.F., Konieczny, A., Cummings, M.P., and Ausubel, F.M.(1990). The structure, distribution and evolution of the TA1 ret-rotransposable element family of Arabidopsis thaliana. Genetics126, 713–721.

Yamamoto, M., and Yamamoto, K. (1998). Differential effects of1-naphthaleneacetic acid, indole-3-acetic acid and 2,4-dichlo-rophenoxyacetic acid on the gravitropic response of roots in anauxin resistant mutant of Arabidopsis, aux1. Plant Cell Physiol. 39,660–664.

DOI 10.1105/tpc.001248; originally published online April 29, 2002; 2002;14;1017-1031Plant Cell

Grierson and Keith LindseyMartin Souter, Jennifer Topping, Margaret Pullen, Jiri Friml, Klaus Palme, Rachel Hackett, Don

Mutants of Arabidopsis Are Defective in Sterol Profiles and Auxin and Ethylene Signalinghydra

 This information is current as of July 16, 2018

 

References /content/14/5/1017.full.html#ref-list-1

This article cites 58 articles, 28 of which can be accessed free at:

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists