The ARG1-LIKE2 Gene of Arabidopsis Functions in a Gravity ... · The ARG1-LIKE2 Gene of Arabidopsis...

The ARG1-LIKE2 Gene of Arabidopsis Functions in aGravity Signal Transduction Pathway That Is GeneticallyDistinct from the PGM Pathway1

Changhui Guan2,3, Elizabeth S. Rosen2,4, Kanokporn Boonsirichai2, Kenneth L. Poff5, andPatrick H. Masson*

Laboratory of Genetics, University of Wisconsin, 445 Henry Mall, Madison, Wisconsin 53706

The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fullygravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxintransport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls displaystrong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1,another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at lowlevels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to thoseof single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic genePHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the singlemutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to thewild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in thesame gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathwayinvolving PGM.

Gravity is one of the environmental cues thatguides plant organs’ growth. Most plant organs arecharacterized by a specific gravity set point angle,which defines their preferential growth vector rela-tive to gravity (Firn and Digby, 1997). In young Ara-bidopsis seedlings, shoots grow upward, displayingnegative gravitropism, whereas roots grow down-ward, toward the center of gravity (positive gravitro-pism; Bullen et al., 1990; Boonsirichai et al., 2002).

Gravity perception by dicot organs involves pri-marily the sedimentation of amyloplasts within spe-cialized cells (statocytes) located in the columellaregion of the root cap and in the starch sheath, which

constitutes the endodermis of hypocotyls and inflo-rescence stems (Kiss et al., 1996; Kuznetsov andHasenstein, 1996; Blancaflor et al., 1998; Weise et al.,2000). In shoots, sedimentable amyloplasts and thecurvature response to gravistimulation occur alongthe elongation zone (for review, see Masson et al.,2002). After amyloplast sedimentation, signals arelikely transduced within the endodermal cells, andphysiological signals are transported laterally to af-fect elongation of cortical and epidermal cells. Inroots, sites of gravity perception and curvature re-sponse may be physically separated (Poff and Mar-tin, 1989). Hence, physiological signals resultingfrom activation of the gravity signal transductionpathway should be transported from the root capcolumella to the elongation zones where the grav-itropic curvature is initiated (for review, see Boonsi-richai et al., 2002).

Auxin is a physiological signal that has been shownto mediate the gravitropic response (for review, seeMasson et al., 2002). In gravistimulated roots, auxinis redistributed asymmetrically across the root capand transmitted to the elongation zones where itpromotes a gravitropic curvature. This redistributionof auxin appears modulated, at least partly, by therelocation of PIN3-containing auxin efflux machin-ery, from a symmetrical distribution along theplasma membrane of columella cells toward an ac-cumulation at their new physical bottom (Friml et al.,2002). This process is accompanied by rapid alkalin-ization of the statocytes’ cytoplasm and acidificationof the cell wall surrounding the statocytes (Scott and

1 This work was supported in part by the National Science Foun-dation (grant nos. MCB–9905675 and MCB–0240084), by the Na-tional Aeronautic and Space Administration (grant nos. NAG2–1336and NAG2–1602 to P.H.M.), by the National Science Foundation/Department of Energy/U.S. Department of Agriculture (TrainingGrant fellowship no. BIR 92–2033 to E.S.R.), and by the Thai Gov-ernment (fellowship to K.B.). This is manuscript no. 3611 from theLaboratory of Genetics, University of Wisconsin (Madison).

2 These authors contributed equally to the paper.3 Present address: Department of Plant Pathology, University of

Wisconsin, 1630 Linden Drive, Madison, WI 53706.4 Present address: Bio-Link North Central Regional Center,

Madison Area Technical College, 3550 Anderson Street, Madison,WI 53706.

5 Present address: Department of Horticulture, Michigan StateUniversity, East Lansing, MI 48824.

* Corresponding author; e-mail [email protected]; fax608 –262–2976.

Article, publication date, and citation information can be foundat http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.023358.

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Allen, 1999; Fasano et al., 2001). Both processes ap-pear essential for full graviresponsiveness.

Polar auxin transport machineries, including auxininflux and efflux carriers that are partly made of theAUX1 and AGR1/EIR1/PIN2/WAV6 gene products,respectively, modulate the transmission of thegravity-induced lateral auxin gradient from the rootcap to the elongation zones (Bennett et al., 1996; Chenet al., 1998; Luschnig et al., 1998; Muller et al., 1998;Utsuno et al., 1998; Swarup et al., 2001). There, anauxin response machinery converts the auxin gradi-ent signal into a differential cellular growth respon-sible for the gravitropic curvature (for review, seeMasson et al., 2002), although an auxin gradient-independent phase may contribute to the early cur-vature response that occurs in the root distal elonga-tion zone (Evans and Ishikawa, 1997; Wolverton etal., 2002).

Little is known about the molecular mechanismsthat underlie gravity signal transduction in root capcells, leading to asymmetric redistribution of auxin.Ca2� may serve as a second messenger in this path-way (Plieth and Trewavas, 2002). On the other hand,experiments carried out in cereal pulvini suggest thatinositol 1,4,5-trisphosphate might also contribute togravity signal transduction, at least in this system(Perera et al., 1999).

Only a few genes have been uncovered that affectgravity signal transduction in roots and hypocotyls.Mutations in two Arabidopsis genes, including SGR2(which encodes a putative phospholipase A1 local-ized in the membranes of vacuoles and small or-ganelles) and ZIG/SGR4 (which encodes the AtVTI11SNARE protein), result in altered hypocotyl andshoot gravitropism, along with misshapen seeds andseedlings (Kato et al., 2002; Morita et al., 2002). Theshoot gravitropic phenotype of sgr2 and zig/sgr4 mu-tants could be rescued by expressing the correspond-ing wild-type genes in the endodermis. These twoproteins may be involved in a vacuolar membranesystem that participates in the early events of gravitysignal transduction specific to shoots (Kato et al.,2002; Morita et al., 2002).

Mutations in the ARG1 gene of Arabidopsis resultin altered root and hypocotyl gravitropism withoutpleiotropic phenotypes. Mutant roots and hypocotylscontain starch in their statocytes and respond likewild type to phytohormones, polar auxin transportinhibitors, and lateral light stimulation (Fukaki et al.,1997; Sedbrook et al., 1999). The ARG1 gene encodesa DnaJ-like protein that carries a coiled-coil domainwith similarity to coiled coils found in several cy-toskeleton binding proteins. Hence, it was postulatedthat ARG1 might mediate gravity signal transductionby promoting the folding, targeting, or degradationof gravitropic regulators in the vicinity of the cy-toskeletal network in statocytes (Sedbrook et al.,1999).

Ninety-one genes encode DnaJ-like proteins inArabidopsis (Arabidopsis Genome Initiative, 2000;Miernyk, 2001). However, phylogenetic studies indi-cate that only two of these genes, named ARG1-LIKE1(ARL1, also called AtDjB16 or At1g24120) and ARG1-LIKE2 (ARL2, also called AtDjC39 or At1g59980), en-code proteins with high-level similarity to ARG1throughout their lengths (Sedbrook et al., 1999; Mi-ernyk, 2001). Nothing is known about the function ofARL1 and ARL2 in plant growth and development. Inthis manuscript, we report on the isolation and phe-notypic characterization of allelic mutations in thesetwo genes.

RESULTS

13-8 Mutation Affects Root and Hypocotyl Gravitropism

To identify Arabidopsis mutants affected in rootand hypocotyl gravitropism, we subjected 103,000fast neutron-mutagenized Estland (Est) seedlings toan on-agar reorientation assay. Three hundred andtwenty four seedlings displayed an altered grav-itropic response. Their progeny were retested foraltered gravitropism, 50 of which again showed adiminished curvature response to gravistimulation.13-8 was one of them. This mutant was backcrossedseven times against the parental wild-type (Est) toremove unlinked mutations.

To better characterize the gravitropic phenotype of13-8, we subjected wild-type and 13-8 mutant seed-lings to an in-agar reorientation assay in darkness.The root and hypocotyl tip angles from the horizon-tal were measured at regular time points after gravi-stimulation. Figures 1A and 2A show that 13-8(arl2-1) mutant roots and hypocotyls displayedslower kinetics of gravitropism compared with wildtype. They also showed increased variation in rootand hypocotyl tip angles compared with wild type ateach time point (F test probabilities below 0.05).

The gravitropic phenotype of 13-8 mutant seed-lings was very similar to that of arg1-2 mutants (Figs.1C and 2A), which displayed no pleiotropic pheno-types (Sedbrook et al., 1999). Therefore, we set upexperiments aimed at determining if 13-8 also af-fected specifically gravitropism, without resulting inpleiotropic phenotypes. Results indicated that 13-8mutant roots grew at wild-type rates in the absenceof phytohormones or polar auxin transport inhibitorsin the medium (Student’s t test probability � 0.48;n � 207–215). They also displayed wild-type rootgrowth sensitivity to auxins indole-3-acetic acid(IAA; Fig. 3A), 1-naphthaleneacetic acid (1-NAA),and 2,4-dichlorophenoxyacetic acid (2,4-D; data notshown), to a polar auxin transport inhibitor (NPA;Fig. 3B), to ACC (a precursor of ethylene biosynthe-sis; Fig. 3C), and to BA (Fig. 3D). In the experimentshown in Figure 3B, a slight but statistically mean-ingful difference in root growth sensitivity to 0.5 �mNPA was observed between wild-type and mutant

ARL2 Functions in Gravity Signal Transduction

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roots (Student’s t test P value of 0.01). However, thisdifference was not reproducible in other experiments(data not shown).

We also tested the mutant’s ability to accumulatestarch in the statocytes. Results indicated that 13-8mutant roots accumulated starch in the columellacells of their root cap similarly to wild-type roots,whereas pgm-1 mutant roots, which are defective inthe phosphoglucomutase enzyme involved in starchbiosynthesis (Caspar and Pickard, 1989), did not(data not shown). Hence, it appears that the 13-8mutation affects root and hypocotyl gravitropism,without altering root growth rate, root growth sensi-tivity to phytohormones or inhibitors of polar auxintransport, or starch accumulation in the statocytes.

To further investigate the possibility that the 13-8mutation might affect more generally the ability ofplant organs to curve in response to environmentalparameters, we characterized the curvature responseof hypocotyls to varying fluences of lateral light stim-ulation (phototropism). Wild-type and 13-8 mutanthypocotyls displayed similar phototropic dose re-sponse curves to lateral light stimulation, with peakresponses at 0.1 �m m�2 radiance. However, 13-8hypocotyls responded more strongly than wild typeto lateral light stimulation at most fluences tested(Fig. 2B; Student’s t test p value of 0.0083 for seed-lings exposed to 0.1 �m m�2 lateral light).

13-8 Affects ARL2, an ARG1 Paralog

The results described above indicated that the 13-8phenotype was similar to that of arg1 mutants (Sed-brook et al., 1999). However, genetic complementa-tion studies suggested that the two mutations werenot allelic, even though both were recessive andmapped on the bottom arm of chromosome 1, be-tween the nga111 and ATHGENEA simple sequencelength polymorphism (SSLP) markers (data notshown; Bell and Ecker, 1994; Sedbrook et al., 1999).Interestingly, one of the ARG1 paralogs, ARL2, islocated within 10 map units of ARG1 on chromosome1 (Arabidopsis Genome Initiative, 2000; Miernyk,2001). Therefore, we decided to determine if the 13-8mutation affects ARL2. We PCR amplified, cloned,and sequenced a piece of genomic DNA surroundingARL2 in wild-type and 13-8 mutant plants. Results

Figure 1. Gravitropic phenotype of wild-type, arl2, and arl1 mutantseedlings in darkness (A and C) or in light (B and D). Average root tipangles from the horizontal are shown at each time point. In A to D,genotypes of tested seedlings are indicated in a legend box. A, Rootgravitropism of wild-type Est, Wassilewskija (Ws), and arl2-1, arl2-2,and arl2-3 mutant seedlings. This graph shows the results of twoindependent experiments, one involving Est and arl2-1, the other oneinvolving Ws, arl2-2, and arl2-3. Wild-type Ws seedlings were in-cluded in both experiments, where they showed almost identicalkinetics of root gravitropism (data not shown). Hence, we show onlythe Ws response for the second experiment. n � 33 to 84 for the firstexperiment and 38 to 120 for the second experiment. B, Root grav-itropism of wild-type Est, untransformed arl2-1 mutant seedlings, and

progeny of two independent arl2-1 transformants carrying thep35S-His6::ARL2 construct (arl2-1[ARL2]3 and arl2-1[ARL2]6, re-spectively; n � 41–53). C, Root gravitropism of wild-type Ws, singlemutants arl1-4, arg1-2, and arl2-1, double mutant arg1-2 arl2-1, andtriple mutant arg1-2 arl2-1 arl1-4 (n � 28–61). D, Root gravitropismof wild type (WT), single mutants arl2-1 and pgm-1, and doublemutant arl2-1 pgm-1 seedlings. All wild-type and mutant lines testedin this experiment were derived from individual segregating F2 prog-eny from a cross between arl2-1 and pgm-1. Two independent arl2-1pgm-1 double-mutant lines (a and b) were analyzed (n � 14–40). InA to D, vertical bars representing SEs are shown at each time point.However, they are often masked by the curve symbols.

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shown in Figure 4A indicated that the 3� end of ARL2is deleted in 13-8 mutant plants as part of an 857-bpdeletion. This deletion eliminates the carboxyl end ofthe predicted protein, including the conserved regionpredicted to form a coiled-coil structure in ARG1 andARL2 (Sedbrook et al., 1999). The next predicted genefound within the same T2K10 bacterial artificial chro-mosome genomic clone (At1g59990) is located 6.7 kbaway from ARL2, on its 3� side (Arabidopsis GenomeInitiative, 2000). Hence, it appeared likely that the13-8 mutation affects the ARL2 gene. This mutationwas named arl2-1.

We used a reverse genetic strategy to screen twocollections of T-DNA-mutagenized plants for indi-

viduals carrying other mutations in ARL2 (Young etal., 2001). Plants carrying two independent T-DNAinsertion alleles of ARL2 were identified and charac-terized. Molecular analysis revealed that arl2-2 con-tains a T-DNA insertion within the predicted ARL2ORF at position 2,550 of the gene (Fig. 4B). Southern-blot analysis confirmed the existence of one T-DNAin this line. However, it also indicated that the ARL2DNA normally present 3� to the T-DNA insertion sitein wild-type DNA was physically separated from theT-DNA insertion and ARL2 5� fragment in this mu-tant (data not shown). Therefore, we used an inversePCR strategy to clone and characterize genomic DNAlocated upstream of the 3� end ARL2 fragment in thisline. Results indicated that this arl2-2 3� end fragmentwas flanked by a 71-bp DNA sequence of unknownorigin (sharing no homologies with sequencespresent in the databases), itself followed by DNAderived from the 3� end of a putative protein phos-phatase 2C gene located on chromosome 2(At2g20050; Fig. 4B). The recombination breakpointwithin At2g20050 was located 537 bp downstream ofthe A residue of the predicted initiation codon. Bothgenes were in opposite orientations within the re-combined structure (Fig. 4B).

To determine if this recombined structure wasstrictly associated with arl2-2, we tested the ability ofAt2g20050-specific PCR primers (PP2C-F1 and PP2C-R2; Table I; Fig. 4B) to amplify At2g20050 DNA over-lapping the recombination breakpoint described inthe previous paragraph from wild-type and arl2-2mutant seedlings. Results shown in Figure 5 indi-cated that these primers allowed amplification of a531-bp fragment from wild-type Ws DNA but notfrom mutant arl2-2 DNA. On the other hand, a 351-bpDNA fragment could be amplified from arl2-2 DNAwhen a combination of ARL2-specific (ARL2-R7) andAt2g20050-specific (PP2C-R2) primers was used inthe reaction (Fig. 5). No fragments could be amplifiedfrom wild-type DNA with this primer pair (Fig. 5).Taken together, these data suggest that arl2-2 re-sulted from the combination of a T-DNA insertionwithin ARL2 and a reciprocal translocation betweenchromosomes 1 and 2 involving breakpoints withinthe ARL2 and At2g20050 genes.

Molecular analysis of arl2-3 revealed that this allelederived from insertion of an 8-kb T-DNA within thefirst predicted intron of the gene. This insertion in-volved a T-DNA flanked by two left borders and wasaccompanied by a small 42-bp deletion of predictedintronic sequences 3� of this insertion site (Fig. 4C).

We isolated homozygous arl2-2 and arl2-3 plantsand tested the gravitropic phenotype of their prog-eny. Results shown in Figure 1A demonstrated thatthe roots of arl2-2 and arl2-3 seedlings display alteredgravitropism, similarly to arl2-1. Mutant hypocotylsalso showed similar alterations in their gravitropicresponse, whereas mutant inflorescence stems dis-

Figure 2. Tropic phenotypes of arl2 shoots. A, Kinetics of hypocotylgravitropism in darkness for wild-type Est, arg1-2, and arl2-1 mutantseedlings (n � 111–168). B, Hypocotyl phototropism of 42-h-oldseedlings exposed to a single pulse of blue light (450 nm) at a fluencerate of 0.15 �M m�2 s�1. Hypocotyl curvatures were measured 30min after phototropic stimulation (n � 58–104). C, Kinetics of pri-mary inflorescence stem gravitropism in darkness for 3- to 3.5-week-old wild-type and arl2-3 mutant plants (n � 15–16). As in Figure 1,SEs are shown by vertical bars that are often masked by the curvesymbols.





played wild-type kinetics of gravitropism (Fig. 2C;data not shown).

A cross between homozygous 13-8 (arl2-1) andarl2-2 plants yielded trans-heterozygous progenythat displayed a gravitropic defect in both roots andhypocotyls. Because these mutations were isolatedindependently in two separate backgrounds (Est andWs) and both were recessive (data not shown), thedata strongly support a role for ARL2 in root andhypocotyl gravitropism. To verify this conclusion,we transformed arl2-1 mutant plants with thep35S-His6::ARL2 construct and analyzed the grav-itropic phenotype of the progeny of two independenttransformants. Results shown in Figure 1B indicatedthat seedlings carrying the transgene developed astronger root curvature response to gravistimulationthan untransformed mutant seedlings or than wild-type seedlings of the corresponding ecotype (Est).The gravitropic defect of arl2-1 hypocotyls was alsorescued by the p35S-His6::ARL2 construct (data notshown).

Null Mutation in ARL1, Another ARG1 Paralog, DoesNot Affect Gravitropism

ARL2 is one of two genes that encode ARG1-likeproteins in Arabidopsis (Fig. 6; Sedbrook et al., 1999;Arabidopsis Genome Initiative, 2000). To investigatethe role of the other ARG1 paralog, named ARL1, inplant growth and development, we isolated the arl1-4mutation, which carries a T-DNA insertion withinthe second predicted intron of ARL1 (Fig. 4D). Al-though ARL1 is expressed in all organs of wild-typeplants (Fig. 7B), no ARL1 mRNA could be detected inhomozygous arl1-4 mutant plants (Fig. 7C), indicat-ing that this mutation is an RNA null. When testedfor gravitropism, homozygous arl1-4 mutant seed-lings displayed wild-type kinetics of root and hypo-cotyl gravitropism, suggesting that ARL1 is not nec-essary for full gravitropism (Fig. 1C). arl1-4 mutantsalso appeared wild type in general plant growth andmorphology and in experiments aimed at testing ger-mination, seedling growth responses to light, phyto-hormones, and polar auxin transport inhibitors (datanot shown). Their roots also appeared to wave likewild type when grown on tilted hard-agar surfaces.Hence, so far, we have not been able to associate aspecific phenotype with the arl1-4 mutation.

ARL2 and ARL1 Encode ARG1-Like Proteins

We cloned full-length ARL2 cDNAs (GenBank ac-cession no. AY226826) by RACE-PCR and sequenced

Figure 3. Root growth sensitivity to phytohormones and a polarauxin transport inhibitor. A to D, Relative root growth rate of wild-type Est (black bars) and arl2-1 mutant seedlings (white bars) in thepresence of varying concentrations of IAA (A), naphthylphthalamicacid (NPA; B), 1-aminocyclopropane-1-carboxylic acid (ACC; C), orN6-benzyladenine (BA; D). Four-day-old seedlings were transferredonto fresh germination medium (GM) containing 0.1% (v/v) ethanol(A and D), 0.05% (v/v) dimethyl sulfoxide (B), or 0.05% (v/v) isopro-panol (C) with the indicated concentrations of IAA, NPA, ACC, or BA.Root growth was measured over a period of 2 d. Average root growthrates were determined for each compound concentration and di-vided by the corresponding growth rate in absence of the compound(control). SEs are represented by vertical bars. The numbers of seed-

lings tested in these experiments were 36 to 52 (A), 27 to 43 (B), 44to 91 (C), and 81 to 95 (D). Average growth rates in the absence ofadded compounds were: A, 5.2 � 0.09 (Est) and 4.5 � 0.08 (arl2-1)mm d�1; B, 4.6 � 0.12 (Est) and 4.5 � 0.12 (arl2-1) mm d�1; C,4.4 � 0.09 (Est) and 5.09 � 0.1 (arl2-1) mm d�1; and D, 5.2 � 0.09(Est) and 5.0 � 0.1 (arl2-1) mm d�1.

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them. Three of four cDNA 5� ends were locatedwithin a 10-bp A-rich region. The fourth transcrip-tion start site was located 53 bp downstream of thefirst one (data not shown). Two polyadenylation siteswere also found at the 3� end, located within 28 bp ofeach other. The first one was located 136 bp down-stream of the translation stop codon (TGA). ARL2contains 10 exons and nine introns. We also clonedARL1 cDNA (GenBank accession no. AY226825) andshowed that ARL1 contains 10 exons and nine in-trons, similarly to ARL2. The position of each intronis conserved within these genes (Fig. 4). It is alsoconserved with the position of introns within ARG1,although the latter gene contains an additional intronwithin its 3� region (Sedbrook et al., 1999). The sev-enth exon of ARL1 and ARL2 was not predicted indatabase annotations of the Arabidopsis genome(At1g24120 and At1g59980).

The first ATG of ARL2 predicted to constitute agood translation initiation site by the Netstart 1.0program (http://www.cbs.dtu.dk/cgi-bin/) is lo-cated 284 bp downstream of the first transcriptionstart site (Fig. 4A). It initiates a large ORF predictedto encode a 415-amino acid polypeptide whose se-quence is similar to that of ARG1 and ARL1 through-out their entire lengths (Fig. 6). All three proteinscontain a J domain at their N terminus, a potentialtransmembrane domain, and a putative coiled-coildomain at the C end (Fig. 6). The arl2-1 deletioneliminates all amino acids downstream of R311, de-leting most of the coiled-coil domain in the protein.The T-DNA insertion and translocation found inarl2-2 disrupts the protein-coding region at T362,whereas the T-DNA insertion in arl2-3 disrupts thecoding potential of ARL2 right at the middle of the Jdomain (Fig. 6).

ARL2 Is Expressed at Low Levels in AllOrgans of the Plant

The ARG1 gene was shown to be expressed ubiq-uitously in Arabidopsis, even though the phenotypicanalysis of arg1 mutants suggested a specific involve-ment in gravity signal transduction (Sedbrook et al.,1999). Similarly, ARL1 is expressed in all organs ofthe plant (Fig. 7B), and its expression is neither af-

Figure 4. Genomic structure of arl2-1 (A), arl2-2 (B), arl2-3 (C), andarl1-4 (D). Exons are represented by rectangles, introns by thin bars,and intergenic chromosomal regions by thick bars. ATG, Translationinitiation codons; TGA or TAG, stop codons, ARL2 and At2g20050 orARL1. Initiation and stop codons for lower strand open readingframes (ORFs; B) are indicated by inverted letters. The nucleotidesequence of a segment of DNA flanking the mutation site in eachallele is indicated at the bottom or top of the corresponding diagram.The nucleotide sequence of an ARL2 or ARL1 exon fragment is

represented in bold and uppercase characters, whereas that of anintronic or intergenic region is represented by uppercase characters.The sequence of At2g20050 exon fragments (B) is represented byitalic uppercase characters. Sequences deleted within a specificallele are represented by lowercase characters, whereas new se-quences added at a specific site (translocation breakpoint betweenthe ARL2 and At2g20050 3�end fragments in arl2-2) are representedby a gray box, and written in bold, italicized, lowercase gray char-acters. T-DNA inserts are represented by arrows. For arl2-2 (B), thetwo ARL2 sequence-containing loci derived from a reciprocal trans-location between chromosomes 1 and 2 are shown, along with theposition of primers (black arrowheads) used in diagnostic PCR reac-tions (Fig. 5).





fected by arl2-1 or arg1-2 mutations nor by arg1-2arl2-1 double mutations (Fig. 7C). To investigate thepattern of ARL2 expression, we extracted RNAs fromstems, rosette and cauline leaves, flowers, and sil-iques of mature plants, and from roots of 3-week-oldliquid culture-grown seedlings, and subjected themto northern-blot analysis, using the ARL2 cDNA as aprobe. Unfortunately, no signal was found using thisprocedure (data not shown). Therefore, we subjectedthe same RNA preparations to reverse transcriptionand RNA Ligase Mediated (RLM)-RACE-PCR. Figure7A shows that ARL2 is expressed in all tissues tested,though at low levels, below the limit of detection bynorthern-blot procedures.

ARG1 and ARL2 May Function in a CommonGenetic Pathway That Is Distinct from the PathwayInvolving PGM

As illustrated in Figures 1, A through D, and 2A,the gravitropic phenotypes associated with muta-

tions in either ARG1 or ARL2 were intermediate be-tween wild-type gravitropism and a complete aboli-tion of the gravitropic response. This afforded anopportunity to genetically test whether the two mu-tations affected parallel branches of the gravity signaltransduction pathway. Homozygous arl2-1 andarg1-2 plants were crossed, and double heterozygousF1 progeny were recovered and shown to developwild-type responses to gravistimulation (data notshown). arg1-2 arl2-1 double mutants were recoveredfrom the segregating F2 progeny, and the gravitropicphenotype of their selfed progeny was analyzed. Fig-ure 1C shows that arg1-2 arl2-1 double mutants dis-played a gravitropic phenotype that was almost iden-tical to the phenotype associated with single mutants.

Using similar procedures, we also isolated arl2-1arl1-4 and arg1-2 arl1-4 double mutants and arl1-4arl2-1 arg1-2 triple mutants. All double and triplemutants displayed kinetics of root gravitropism thatwere similar to those of single mutants (Fig. 1C; datanot shown).

Because ARG1, ARL2, and PGM all appear to func-tion in gravity perception and/or signal transduction(Kiss et al., 1989; Sedbrook et al., 1999), we alsogenerated arl2-1 pgm-1 double mutants and tested thegravitropic phenotype of their progeny. Resultsshown in Figure 1D demonstrated that arl2-1 pgm-1double mutant roots displayed much stronger grav-itropic defects than single mutants or wild-typeroots. Furthermore, the population of arl2-1 pgm-1roots displayed enhanced deviation from the verticalcompared with wild-type or single mutant popula-tions in the absence of gravistimulation (Fig. 1D; Ftest P values below 0.05). Hypocotyls of the doublemutants behaved similarly (data not shown).

Figure 5. arl2-2/At2g20050 recombinant DNA fragments can bePCR amplified from arl2-2 genomic DNA but not from wild-typeDNA. Primers used to PCR amplify wild-type ARL2 or recombinantarl2-2/At2g20050 fragments from Ws or arl2-2 DNAs are shown byhorizontal arrowheads on the diagram shown in Figure 4B andmarked F1 (PP2C-F1), R2 (PP2C-R2), XR2, and R7 (ARL2-R7) on thatdiagram and at the top of the gel. Their respective sequences areprovided in Table I. The DNA used as a template in diagnostic PCRreactions is indicated above each electrophoretic lane. The sizes ofamplified fragments, as determined by comparison with Mr markersloaded on the same gel, are shown on the right of the gel.

Table I. Primers used for PCR amplifications of ARG1, ARL1, andARL2 sequences

All primers were ordered from Integrated DNA Technologies, Inc.(Coraville, IA).

Primer Name Primer Sequence

ARG1-F 5�-ATCCTCCTTTCTTCATCGTTTCTT-3�ARG1-R 5�-CATTGTCATAGTGCCTTCTCTTTT-3�ARL1-F1 5�-TTCAATAGGGCACAATATGTTTAAGAGAACT-3�ARL1-F4 5�-GGAGGTCACGTTTTCTTACAAC-3�ARL1nestr1 5�-CAATTTCCGGTAAGCGCTC-3�ARL1nestr2 5�-GACACAGCTTGTCCAAGTAC-3�ARL1-R1 5�-CCAAGTCGGAGAAGAAGGATGCTGATAAG-3�ARL2-F 5�-GGAGAATAAAGACGCCGGAGAAGAAGATGA-3�ARL2-F6 5�-TGGTCTATGGTTTATCTCTTTGCACACTTTG-3�ARL2-F7 5�-CACACTTTGCCTGATTCATCTCTTGCTA-3ARL2GFP2f2 5�-TCCCCCGGGATGGCCACTCATTCATC-3�ARL2KOFU3 5�-GGACAAACTGGCTGTAGGGAAAGA-3�ARL2KOFL1 5�-CGAAACGATAAACAGGGAAGCCGA-3�ARL2-R 5�-GTAAGCCGCACATATCTCATTCCTCCTCT-3�ARL2-R2 5�-TAACTAAACCAACAAAACCAAAGGGAT-3�ARL2-r3 5�-CAGCTCCTAAGCTTGACAG-3�ARL2-R6 5�-ATATTGTACCATTTCTTCTTCTTGACTCCA-3�ARL2-R7 5�-CACCTTCTTCCCTAACTGCGACTTCTCCTTC-3�ARL2-SL2 5�-TTCAACGCCTCCCCCAATAAAT-3�ARL2-SU1 5�-AGCTCATCTTCTTCTCCGGCGTCTTTATT-3�ARL2-SU3 5�-GCTGGTTCTCATGTCTTTGCTGTT-3�GFPPAr 5�-CATGCTTAACGTAATTCAAC-3�6HisOEf 5�-TGCCATGGATGAACTATACAAAG-3�JL202 5�-CATTTTATAATAACGCTGCGGACATCTAC-3�JL270 5�-TTTCTCCATATTGACCATCATACTCATTG-3�PP2C-F1 5�-GATGCGGTGGATGATGTAGATAACGACGAA-3�PP2C-R2 5�-AGCTAAAACCGCCCTGGAATC-3�pCB35SPf 5�-GCCTTCAGTTGAGCTCCATGG-3�pCB6Hisr2 5�-CTGGTCACCTCTAGACACGTG-3�pCBNOSPAf 5�-CCGATCGCTCGAGCATTTGGC-3�pCBNOSPAr 5�-CACTGGTACCTTAATTCCCGA-3�T3 5�-AATTAACCCTCACTAAAGGG-3�T7 5�-AATACGACTCACTATAG-3�XR2 5�-TGGGAAAACCTGGCGTTACCCAACTTAAT-3�

Guan et al.




DISCUSSION

In this paper, we demonstrate that three indepen-dently isolated mutations in the ARL2 locus, whichencodes a DnaJ-like protein similar to ARG1 (Sed-brook et al., 1999), affect root and hypocotyl gravitro-pism of seedlings grown under light (Fig. 1, B and D)or darkness (Figs. 1, A and C, and 2A). All threemutations are recessive, and do not complement eachother. We also show that the gravitropic phenotypeassociated with arl2-1 can be rescued by transforma-tion with a construct that drives expression of aHis-tagged version of the ARL2 protein (Fig. 1B).Together, these data demonstrate that ARL2 is in-volved in root and hypocotyl gravitropism inArabidopsis.

The agravitropic phenotype associated with arl2-1and arl2-3 is stronger than that associated with arl2-2(Fig. 1A). This difference could reflect the relativestrengths of these mutations. arl2-1 carries a 3� dele-tion that eliminates most of a predicted coiled-coildomain at the C terminus of the ARL2 protein, po-tentially eliminating its ability to interact with targetproteins. Similarly, arl2-3 carries a T-DNA insertwithin its first intron, possibly disrupting the coding

potential of this gene within the J domain. These twoalleles are likely to be null, although the low level ofARL2 expression in wild-type plants precludes care-ful verification of this prediction. arl2-2, on the otherhand, contains a T-DNA insertion near the 3� end ofthe gene’s ORF, downstream of its coiled-coil-encoding domain (Figs. 4 and 6). Hence, unlike arl2-1and arl2-3, arl2-2 could potentially encode a func-tional protein that may interact with its targets.

The arl2-2 mutation involved a complex recombi-nation event that also disrupted a putative proteinphosphatase 2C gene (At2g20050). In this line, the 3�end of At2g20050 is associated with the 3� end ofarl2-2 and a 71-bp insertion of unknown origin. Asecond locus carrying the 5� end of ARL2 associatedwith a T-DNA and the complementary 5� end ofAt2g20050 was also found in this line (Figs. 4B and 5).No wild-type ARL2 or At2g20050 could be found inthis mutant, strongly suggesting that this recombina-tion involved a reciprocal translocation betweenchromosomes 1 and 2, with breakpoints located atpositions 2,550 of ARL2 and 537 of At2g20050 (Fig.4B). It is not unusual to find chromosomal rearrange-ments associated with T-DNA insertions in plants

Figure 6. Amino acid sequence alignments of the ARG1 (top), ARL1 (middle), and ARL2 (bottom) proteins (Ws ecotype).Amino acid positions within each protein are shown at the left of each sequence (brown numbers). Protein names areindicated at the left of these numbers. Amino acids that are identical or conserved between at least two of the three proteinsare shown in red and green characters, respectively. Gaps introduced within a protein to allow better alignment of flankingsequences are represented by dashes. The J domain, putative transmembrane domain, and predicted coiled-coil region areboxed. The arl2-1 mutation deletes the doubly underlined (orange) carboxyl end of the protein, whereas positions of T-DNAinserts within ARL2 and ARL1 alleles are indicated by black and blue arrows, respectively. Alignments were obtained by theBoxshade server (Institut Pasteur, Paris; http://bioweb.pasteur.fr/seqanal/interfaces/boxshade.html).





(Tax and Vernon, 2001). Unfortunately, the complexnature of this arl2-2 mutation also implies that thephenotypes found in this line could be associatedwith alterations in ARL2, in At2g20050, or in bothgenes. It is possible that the mutation in the putativeprotein phosphatase 2C At2g20050 gene could con-tribute to the gravitropic phenotype found in thisline. For instance, mutations in RCN1, which encodesa protein phosphatase 2A subunit, also affect grav-itropism (Rashotte et al., 2001). However, it is likelythat the T-DNA insertion and 3� end translocationfound in arl2-2 also contribute to the gravitropic de-fect in this line. F1 progeny from a cross between

homozygous arl2-1 and arl2-2 plants display a rootgravitropism defect (data not shown).

The hypothesis that mutations in ARL2 are respon-sible for the gravitropic phenotype found in thesemutant lines is supported by the results of transfor-mation experiments in which arl2-1 mutant seedlingswere transformed with the p35S-His6::ARL2 con-struct. The transgenic progeny of independent trans-formants displayed a strong root gravitropic re-sponse relative to untransformed arl2-1 seedlings(Fig. 1B). It is interesting to note, however, that arl2-1roots overexpressing ARL2 displayed enhanced grav-itropism compared with wild-type Est roots (Fig. 1B).This result suggests that ARL2, or a component of theARL2 pathway, might be limiting for the gravitropicresponse in the Est background.

Loss-of-function mutations in ARL2 affect root andhypocotyl gravitropism without altering inflores-cence stem gravitropism (Figs. 1A and 2, A and C).This result is in agreement with previous observa-tions indicating that the gravitropic responses of dif-ferent organs are genetically separable (Bullen et al.,1990). Interestingly, arl2 mutations do not affect rootgrowth rate, root growth resistance to phytohor-mones and polar auxin transport inhibitors, or starchaccumulation in root statocytes. Hypocotyl phototro-pism, on the other hand, is slightly enhanced in thearl2-1 mutant compared with the wild type, at leastwithin the first positive phase of the response (Fig.2B). This slight enhancement of phototropism may bethe consequence of the gravitropic defect associatedwith this mutation. In wild-type seedlings, gravitro-pism interferes with phototropism in directing organtip curvature in response to lateral light stimulation(Correll and Kiss, 2002). In any case, the physiologi-cal data shown here strongly support a role for ARL2in early phases of root and hypocotyl gravitropism,possibly in gravity signal transduction.

It is interesting to note that ARG1, an ARL2 para-log, was also proposed to function in gravity signaltransduction, based on a similar combination of phe-notypes (Sedbrook et al., 1999; K. Boonsirichai andP.H. Masson, unpublished data). Therefore, we ana-lyzed the gravitropic defect associated with arg1-2arl2-1 double mutants. As expected, arg1-2 arl2-1double mutants displayed a gravitropic defect thatwas not exacerbated compared with single mutants(Fig. 1C). This result supports an involvement ofARG1 and ARL2 in the same genetic pathway.

Our analysis of arl2-1 pgm-1 root gravitropism re-vealed a strong phenotypic enhancement comparedwith single mutants (Fig. 1D). Similar observationswere recently made with arg1-2 pgm-1 double mu-tants (K. Boonsirichai and P.H. Masson, unpublisheddata). These striking results strongly suggest thatARL2/ARG1 and PGM function in distinct branchesof the gravity signal transduction pathway. Severalphysiological data support the existence of a second-ary gravity perception mechanism, in addition to

Figure 7. The ARL2 and ARL1 genes are expressed ubiquitously inArabidopsis seedlings and plants. A, 3� ARL2 cDNA fragments can bereverse transcription-PCR amplified from mRNAs extracted from sil-iques (1), cauline leaves (3), rosette leaves (4), stems (5), and flowers(6) of mature plants, roots of 3-week-old liquid-grown plants (7), andcotyledon and leaves of 5-d-old seedlings (2). B, Northern-blot anal-ysis of total RNAs extracted from the plant organs defined in A, usingARL1 cDNA as a probe. Twenty micrograms of total RNA was loadedin each lane. C, Northern-blot analysis of total RNAs extracted fromarl1-4, arl2-1, arg1-2 arl2-1, arg1-2, and wild-type Ws seedlings,using ARL1 (upper) or eIF4A (lower; loading control) cDNA se-quences as probes. In this experiment, the ARL1 probe detected alow-intensity, nonspecific signal in all RNAs tested (including arl1-4).However, the specific ARL1 signal was not detectable in RNAsextracted from arl1-4 seedlings. In each panel, the source of mRNAis indicated above each lane, whereas the probe is identified at theright of the panel.

Guan et al.




amyloplast sedimentation within the statocytes (Ish-ikawa and Evans, 1990; Blancaflor et al., 1998; Fasanoet al., 2001; Wolverton et al., 2002), and it is possiblethat ARL2 and ARG1 function in this alternativepathway. Alternatively, ARL2 could directly or indi-rectly regulate the activity of specific step(s) in thegravity signal transduction pathway controlled byamyloplast sedimentation in the statocytes. Experi-ments are underway to test these models.

As components of HSP70-containing macromolec-ular chaperone complexes, DnaJ-like proteins inter-act with HSP70 through their J domain and withspecific targets through a more divergent proteininteraction domain (Zuber et al., 1998; Miernyk,2001). Because the coiled-coil domain of ARG1 dis-plays similarity to coiled coils found in a number ofproteins that bind to cytoskeletal elements, we pre-viously postulated that ARG1 might interact with thecytoskeleton, or modulate the formation of macromo-lecular signal transducing complexes in vicinity ofthe cytoskeleton (Sedbrook et al., 1999). Recent bio-chemical and immunocytological experiments ap-pear to support this model (K. Boonsirichai and P.H.Masson, unpublished data). Because ARG1 andARL2 share substantial sequence similarity withinthe putative coiled-coil region (Fig. 6), we hypothe-size that ARL2 might also function in gravitropismthrough some interaction with cytoskeletal elements.Current work focuses on identifying and character-izing proteins that interact physically or geneticallywith the C-terminal ends of ARG1 and/or ARL2 andstudying their involvement in gravitropism.

Like ARG1, ARL2 is expressed in all tissues of theplant, though at much lower levels (Fig. 7). Thisresult appears to contradict a model postulating aspecific involvement of ARL2 in gravity signal trans-duction. However, as previously discussed for ARG1(Sedbrook et al., 1999), ARL2 may be involved inother processes that we have not yet analyzed. Alter-natively, it is possible that other ARL2 functions aremasked by functional redundancy.

Finally, we want to emphasize that not all dnaJ-likeproteins are needed for root gravitropism. The Ara-bidopsis genome contains 91 genes encoding DnaJ-like proteins, many more than any other organismwhose genome has now been sequenced (Miernyk,2001; data not shown). Among these DnaJ-like pro-teins, only ARL2 and ARL1 are highly similar toARG1 along their entire lengths (Fig. 6). Interest-ingly, an RNA-null allele of ARL1 (arl1-4) did notaffect root or hypocotyl gravitropism (Fig. 1C; datanot shown). Furthermore, incorporation of arl1-4 inarg1-2 and/or arl2-1 mutant backgrounds did notaffect their gravitropic phenotype (Fig. 1C; data notshown). Therefore, ARL1 does not appear to functionin gravitropism, and experiments are underway todefine its function in plant growth, development, orresponse to the environment.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

To generate a collection of fast neutron-mutagenized seeds, approxi-mately 250,000 seeds of the Est ecotype were exposed to 6,000-Rad fast-neutron radiation by Dr. H. Brunner (Plant Breeding Unit, InternationalAtomic Energy Agency, Vienna). Mutagenized M1 seeds were separatedinto 29 subpopulations. M2 seeds were harvested in bulk for each subpopu-lation and examined separately in the gravitropism mutant screen describedbelow.

T-DNA insertional mutants were obtained through the ArabidopsisKnockout Facility (University of Wisconsin Biotechnology Center, Madison)from populations of T-DNA-transformed Arabidopsis plants (Ws ecotype;Krysan et al., 1999). pgm-1 mutant seeds (Columbia ecotype) were obtainedfrom Tim Caspar (DuPont Co., Wilmington, DE).

All plant manipulations, including surface sterilization of seeds, platingon agar-based media, growth conditions, transplantation of seedlings fromplates to soil, pollination, and harvesting were as previously described(Rutherford and Masson, 1996; Sedbrook et al., 1999). GM contained one-half-strength Murashige and Skoog salts with 1.5% (w/v) Suc and macro-and micronutrients (Invitrogen, Carlsbad, CA).

Quantification of Root and Hypocotyl Gravitropism

Gravitropic Reorientation Assay

Approximately 30 to 80 surface-sterilized seeds were either embedded inor placed on the surface of GM medium containing 0.8% (w/v) agar, inin-agar or on-agar reorientation assays, respectively. Plated seeds wereplaced for 3 to 5 d in darkness at 4°C and germinated vertically at 22°C for2 to 3 d, and then rotated 90°, as described (Sedbrook et al., 1999). Pictureswere taken at regular time intervals (defined in the text) with a COOL-PIX800 Digital Camera (Nikon Inc., Melville, NY).

To quantify the gravitropic response of primary inflorescence stems,wild-type Ws and arl2-3 mutant plants were grown in soil for 3 to 3.5 weeks.At that age, the young plants carried 4- to 8-cm-long primary inflorescencestems. They were positioned horizontally in a dark growth chamber, and theinflorescence stems were photographed at regular time intervals, as de-scribed above.

Root, hypocotyl, and inflorescence stem curvature responses to gravis-timulation were determined on digital images using the National Institutes ofHealth Image Analysis Software version 1.62 (http://rsb.info.nih.gov/nih-image/).

Root Wave Assay

Root waving on tilted surfaces were tested as described in Rutherfordand Masson (1996).

Quantification of Hypocotyl Phototropism

Hypocotyl phototropism was characterized using the procedures de-scribed in Steinitz and Poff (1986). Dark-grown seedlings were exposed to asingle pulse of 450-nm blue light at the fluence rate of 0.15 �m m�2 s�1,provided by a projector equipped with a 300-W ELH tungsten halogen lamp(Osram Sylvania, Inc., Danvers, MA) and a 450-nm interference filter with ahalf bandwidth of 10 nm (PTR Optics, Waltham, MA). The duration of lightexposure was adjusted to generate a fluence response curve in the range offirst positive phototropism, using a Uniblitz shutter (Vincent Associates,Rochester, NY; Janoudi and Poff, 1990). After the curvature was allowed todevelop for 30 min in darkness, seedlings were mounted on clear tape, andanalyzed as described (Steinitz and Poff, 1986).

Analysis of Root Growth Sensitivity toPhytohormones and Polar Auxin Transport Inhibitors

Assays aimed at testing root growth in the presence of phytohormones orpolar auxin transport inhibitors were carried out as described by Sedbrooket al. (1999). IAA, 2,4-D, 1-NAA, ACC, and BA were obtained from Sigma





(St. Louis). NPA was obtained from Pfaltz & Bauer, Inc. (Waterbury, CT).Stock solutions were prepared in ethanol (IAA, 2,4-D, 1-NAA, and BA),isopropanol (ACC), and dimethyl sulfoxide (NPA). All compounds wereadded to the pH-buffered medium at the appropriate concentrations, andthe solvent concentrations were corrected to the same value for all plates ineach assay.

Statistical Analysis of the Data

Data derived from gravitropic and phototropic assays, and root growthresponses to added compounds were subjected to statistical analysis (Excel,Microsoft Corporation, Redmond, WA), using Student’s t tests to comparemean values between wild-type and mutant populations and F tests tocompare deviations from the mean value between genotypes. Differenceswere assumed to be insignificant when the P values associated with thesetests exceeded 0.05.

Detection of Starch within the Columella Cells ofWild-Type and Mutant Root Caps

Seedlings were grown on vertically oriented 0.8% (w/v) agar-based GMmedium in the light for 3 d. They were then grown for 1 more d in darkness,before being exposed for 3 h to an I2:KI solution (Sedbrook et al., 1999). Theywere cleared for 1 h in Hoyer’s solution (Newman et al., 2002) and analyzedas described by Sedbrook et al. (1999).

Mapping of arl2-1

Two mapping populations were generated by crossing arl2-1 mutantplants with wild-type plants of the Landsberg erecta or Columbia ecotypes,respectively. Their progeny were propagated by self-fertilization. F3 prog-eny were subjected to a root wave assay to determine their gravitropicphenotype. This progeny typing allowed a determination of the ARL2genotype of the corresponding F2 parent. Two pools of F3 progeny derivedfrom 15 homozygous wild-type and 15 homozygous mutant F2 plants,respectively, were generated and used to map arl2-1 by bulked segregantanalysis (Michelmore et al., 1991). DNA was extracted from each pool andsubjected to PCR amplification, using primer pairs from the MapPairs kit(Research Genetics Inc., Huntsville, AL). Linkage between ARL2 and spe-cific SSLPs was confirmed by typing the linked SSLPs in individual F3

families.

Molecular Cloning and Characterization of ARL2

DNA and RNA Isolation

Large-scale isolation of total DNA from plants was accomplished asdescribed by Dellaporta et al. (1985). Genomic DNA for PCR amplificationwas isolated from individual cotyledons (Klimyuk et al., 1993), whereasRNA was extracted from seedlings or plant organs (Chang et al., 1993).

Basic Molecular Biology Procedures

Enzymes used in the manipulation of DNA were purchased from NewEngland Biolabs (Beverly, MA). Recombinant DNA techniques, randompriming to radiolabel the DNA, Southern- and northern-blot analyses, andPCR amplifications were carried out as described by Ausubel et al. (1994).DNA sequencing was carried out by the University of Wisconsin Biotech-nology Center DNA Sequencing Facility (Madison).

Cloning of ARL2 Genomic and cDNA Sequences

A piece of genomic DNA carrying the predicted ARL2 gene (At1g59980:Arabidopsis Genome Initiative, 2000) was PCR amplified from Ws genomicDNA, using the ARL2-F and ARL2-R primers (Table I), the ExTaq polymer-ase (TaKaRa Shuzo Co., LTD, Otsu, Japan), and thermocycle conditionsdictated by the Lasergene PrimerSelect program (DNASTAR Inc., Madison,WI). A PCR amplification approach was also used to clone ARL2 cDNAfrom the CD4-6 (flower cDNA), CD4-12 (silique cDNA), and CD4-22 (cDNA

from 3-day-old etiolated seedlings) cDNA phage libraries, obtained fromthe Arabidopsis Biological Resource Center (Ohio State University, Colum-bus), using T7 and T3 primers along with either the ARL2-F or ARL2-Rprimers (Table I). PCR-amplified genomic and cDNA products were clonedinto a pZERO vector (Invitrogen) and sequenced.

The 5� end of the ARL2 mRNA was mapped on the sequenced genomicDNA using the Ambion First-Choice RLM-RACE PCR kit, as recommendedby the supplier (Ambion, Inc., Austin, TX). Primers ARL2-SL2 and ARL2-r3(Table I) were used in these assays, along with the Ambion kit primers, innested PCR amplifications. PCR-amplified cDNAs were cloned into pBlue-script (Stratagene Co., Cedar Creek, TX), and sequenced.

A 3�-RACE procedure was also used to detect ARL2 transcripts in totalRNAs extracted from plant organs (Chang et al., 1993), using primersARL2-SU1 and ARL2-F6 (Table I), along with Ambion First-Choice RLM-RACE PCR kit primers.

Identification and Characterization of arl2-2 and arl2-3

Two T-DNA-insertional mutants (arl2-2 and arl2-3; Ws ecotype) wereisolated through the Arabidopsis Knockout Facility, using the T-DNA-specific JL202 and ARL2-specific ARL2KOFU3 and ARL2KOFL1 primers(Table I; Krysan et al., 1999). PCR-amplified T-DNA-flanking genomic frag-ments were sequenced. arl2-2 was derived from the ALPHA T-DNA collec-tion, whereas arl2-3 was isolated from the BASTA collection(http://www.biotech.wisc.edu/Arabidopsis/).

To investigate the structure of arl2-2, DNA was isolated from homozy-gous plants, cleaved with NsiI or BglII, and subjected to Southern-blotanalysis. Membranes were hybridized successively with PCR-amplifiedARL2-specific probes corresponding to sequences flanking the arl2-2T-DNA on the 5� side, on the 3� side, and to neomycin phosphotransferaseII- or �-glucuronidase-specific T-DNA probes (Rutherford et al., 1998).

Genomic DNA associated with the arl2-2 3� end segment was isolated byinverse PCR of HindIII-digested genomic DNA after self-ligation (Ochmanet al., 1988). Nested PCR amplification was performed to amplify ARL2-flanking sequences, using the ARL2-F6 and ARL2-R6 primers, and then theARL2-F7 and ARL2-R7 primers (Table I). The Elongase DNA polymerase(Invitrogen) was used in these reactions. PCR products were sequenced.

To characterize the arl2-3 mutation, arl2-3 genomic DNA was cleavedwith NcoI and NdeI and subjected to Southern-blot analysis using ARL2cDNA and T-DNA left-border probes (Ausubel et al., 1994). The analysisallowed us to verify that arl2-3 plants contain only one T-DNA insert andthat this T-DNA disrupts the ARL2 gene (data not shown).

Complementation of arl2-1

To verify that the gravitropic defect is truly associated with mutations inARL2, we cloned a His-tagged ARL2 cDNA into the pCAMBIA1302 binaryvector (CAMBIA, Canberra City, Australia), under the control of the CaMV35S promoter and NOS terminator sequences. This construct was generatedas follows. The 35S::GFP::His6 DNA cassette carried by pCAMBIA1302 wasPCR amplified with primers pCB35SPf and pCB6Hisr2 (Table I) and clonedbetween the SacI and XbaI sites of pBluescript II KS(�). A NOS terminatorsequence was then amplified from pCAMBIA1302, using primers pCBNO-SPAf and pCBNOSPAr (Table I), and inserted into this plasmid, between theXhoI and KpnI sites, generating the nGFP2a plasmid. The ARL2 cDNA,obtained by PCR amplification with the ARL2GFP2f2 and T3 primers (TableI), was inserted between the EcoRI and SmaI sites of nGFP2a. A His6::ARL2cassette was amplified from this recombinant plasmid with primers6HisOEf and GFPPAr (Table I), digested with NcoI and EcoRV, and clonedinto pCAMBIA1302, between the NcoI and PmlI sites, thereby replacing theoriginal GFP::His6 cassette of this plasmid. The resulting plasmid was calledp35S-His6::ARL2.

We introduced the p35S-His6::ARL2 construct in wild-type and mutantplants by in planta transformation (Bent, 2000). Transformants were isolatedon GM plates containing 20 �g mL�1 hygromycin (Calbiochem, San Diego)and 0.8% (w/v) agar, transferred to soil, and allowed to self-pollinate. Theprogeny of two of these transgenic plants were subjected to an in-agarreorientation assay under constant light. Individual seedlings carrying aT-DNA insert were identified by PCR amplification, using the 6HisOEf andGFPPAr primers (Table I), and root gravitropism was quantified on theseseedlings.

Guan et al.




Molecular Cloning and Characterization of ARL1

Procedures used to clone and characterize the ARL1 cDNA were asdescribed above for ARL2, except that primers ARL1-F1 and ARL1-R1 (TableI) were used in the PCR reactions, along with T3 or T7 primers (Table I). The5� end of the ARL1 cDNA was mapped on the genomic DNA by RLM-RACEamplification, using the ARL1nestr2 and ARL1nestr1 primers along withAmbion First-Choice RLM-RACE PCR kit primers (Table I; Ambion, Inc).

The arl1-4 allele was identified in the BASTA collection of T-DNA inser-tion lines (Arabidopsis Knockout Facility), using the ARL1-F1 and T-DNAleft-border (JL202) primers. Amplified DNA was subjected to nested PCRwith JL270 and ARL1nestr2 and sequenced.

ARL1 expression was analyzed by northern-blot analysis of total RNAextracted from seedling and mature plant organs (Fig. 7), using 32P-labeledARL1 cDNA as a probe (Ausubel et al., 1994).

Analysis of Possible Genetic Interactions betweenarg1-2, arl1-4, arl2-1, and pgm-1

Plants carrying both the arl2-1 and either arg1-2 or pgm-1 mutations at thehomozygous state were obtained by crossing single mutants and selfing thecorresponding F1 progeny. A cotyledon was excised from each F2 plant andprepared for PCR amplification (Klimyuk et al., 1993). The arg1-2 frameshiftmutation destroys a BamHI restriction site within the ARG1 locus (Sedbrooket al., 1999), creating a BamHI RFLP that can be resolved by BamHI digestionof PCR-amplified ARG1 product (ARG1-F and ARG1-R primers; Table I). Onthe other hand, the ARL2-SU3 and ARL2-R2 primers were used to PCRamplify the ARL2 sequence that surrounds the 857-bp deletion present inarl2-1. Homozygosity at ARL2 was confirmed by a Southern-blot analysis.Plants homozygous for pgm-1 were identified by their starchless phenotype,using the I2:KI staining protocol described above. F2 seedlings with thespecified genotype were grown and selfed. Their progeny were subjected toan in-agar reorientation assay.

arg1-2 arl2-1 arl1-4 triple mutants were obtained by crossing an arg1-2arl2-1 double mutant with an arl1-4 single mutant and selfing the corre-sponding F1 progeny. F2 plants were genotyped as described above for thepresence of arg1-2 and arl2-1 at the homozygous state. PCR amplificationwas also used to identify the arl1-4 allele in DNA extracted from individualcotyledons, using primers ARL1-nestr2 and JL270. Presence of the wild-typeARL1 allele was analyzed by PCR amplification, using the ARL1-F4 andARL1-nestr2 primers. Individual F2 plants with a specified genotype weregrown and selfed. Their progeny were subjected to an in-agar reorientationassay.

ACKNOWLEDGMENTS

We thank the Arabidopsis Knockout Facility (University of Wisconsin,Madison) for providing the arl1-4, arl2-2 and arl2-3 mutants. We also thankJessica Will and Nicole Ammerman for excellent technical assistance.

Received March 11, 2003; returned for revision April 8, 2003; accepted May22, 2003.

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