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First published online July 24, 2003; 10.1104/pp.103.023358 Plant Physiology 133:100-112 (2003) © 2003 American Society of Plant Biologists The ARG1-LIKE2 Gene of Arabidopsis Functions in a Gravity Signal Transduction Pathway That Is Genetically Distinct from the PGM Pathway1Laboratory 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 fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong 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 low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.
Gravity is one of the environmental cues that guides plant organs' growth. Most plant organs are characterized by a specific gravity set point angle, which defines their preferential growth vector relative to gravity (Firn and Digby, 1997  ). In young Arabidopsis seedlings, shoots grow upward, displaying negative gravitropism, whereas roots grow downward, toward the center of gravity (positive gravitropism; Bullen et al., 1990; Boonsirichai et al., 2002).
Gravity perception by dicot organs involves primarily the sedimentation of amyloplasts within specialized cells (statocytes) located in the columella region of the root cap and in the starch sheath, which constitutes the endodermis of hypocotyls and inflorescence stems (Kiss et al., 1996
Auxin is a physiological signal that has been shown to mediate the gravitropic response (for review, see Masson et al., 2002
Polar auxin transport machineries, including auxin influx and efflux carriers that are partly made of the AUX1 and AGR1/EIR1/PIN2/WAV6 gene products, respectively, modulate the transmission of the gravity-induced lateral auxin gradient from the root cap to the elongation zones (Bennett et al., 1996
Little is known about the molecular mechanisms that underlie gravity signal transduction in root cap cells, leading to asymmetric redistribution of auxin. Ca2+ may serve as a second messenger in this pathway (Plieth and Trewavas, 2002
Only a few genes have been uncovered that affect gravity signal transduction in roots and hypocotyls. Mutations in two Arabidopsis genes, including SGR2 (which encodes a putative phospholipase A1 localized in the membranes of vacuoles and small organelles) and ZIG/SGR4 (which encodes the AtVTI11 SNARE protein), result in altered hypocotyl and shoot gravitropism, along with misshapen seeds and seedlings (Kato et al., 2002
Mutations in the ARG1 gene of Arabidopsis result in altered root and hypocotyl gravitropism without pleiotropic phenotypes. Mutant roots and hypocotyls contain starch in their statocytes and respond like wild type to phytohormones, polar auxin transport inhibitors, and lateral light stimulation (Fukaki et al., 1997
Ninety-one genes encode DnaJ-like proteins in Arabidopsis (Arabidopsis Genome Initiative, 2000
13-8 Mutation Affects Root and Hypocotyl Gravitropism To identify Arabidopsis mutants affected in root and hypocotyl gravitropism, we subjected 103,000 fast neutron-mutagenized Estland (Est) seedlings to an on-agar reorientation assay. Three hundred and twenty four seedlings displayed an altered gravitropic response. Their progeny were retested for altered gravitropism, 50 of which again showed a diminished curvature response to gravistimulation. 13-8 was one of them. This mutant was backcrossed seven times against the parental wild-type (Est) to remove unlinked mutations. To better characterize the gravitropic phenotype of 13-8, we subjected wild-type and 13-8 mutant seedlings to an in-agar reorientation assay in darkness. The root and hypocotyl tip angles from the horizontal were measured at regular time points after gravi-stimulation. Figures 1A and 2A show that 13-8 (arl2-1) mutant roots and hypocotyls displayed slower kinetics of gravitropism compared with wild type. They also showed increased variation in root and hypocotyl tip angles compared with wild type at each time point (F test probabilities below 0.05).
The gravitropic phenotype of 13-8 mutant seedlings was very similar to that of arg1-2 mutants (Figs. 1C and 2A), which displayed no pleiotropic phenotypes (Sedbrook et al., 1999
We also tested the mutant's ability to accumulate starch in the statocytes. Results indicated that 13-8 mutant roots accumulated starch in the columella cells of their root cap similarly to wild-type roots, whereas pgm-1 mutant roots, which are defective in the phosphoglucomutase enzyme involved in starch biosynthesis (Caspar and Pickard, 1989 To further investigate the possibility that the 13-8 mutation might affect more generally the ability of plant organs to curve in response to environmental parameters, we characterized the curvature response of hypocotyls to varying fluences of lateral light stimulation (phototropism). Wild-type and 13-8 mutant hypocotyls displayed similar phototropic dose response curves to lateral light stimulation, with peak responses at 0.1 µM m-2 radiance. However, 13-8 hypocotyls responded more strongly than wild type to lateral light stimulation at most fluences tested (Fig. 2B; Student's t test p value of 0.0083 for seedlings exposed to 0.1 µM m-2 lateral light).
The results described above indicated that the 13-8 phenotype was similar to that of arg1 mutants (Sedbrook et al., 1999
We used a reverse genetic strategy to screen two collections of T-DNA-mutagenized plants for individuals carrying other mutations in ARL2 (Young et al., 2001 ). Plants carrying two independent T-DNA insertion alleles of ARL2 were identified and characterized. Molecular analysis revealed that arl2-2 contains a T-DNA insertion within the predicted ARL2 ORF at position 2,550 of the gene (Fig. 4B). Southern-blot analysis confirmed the existence of one T-DNA in this line. However, it also indicated that the ARL2 DNA normally present 3' to the T-DNA insertion site in wild-type DNA was physically separated from the T-DNA insertion and ARL2 5' fragment in this mutant (data not shown). Therefore, we used an inverse PCR strategy to clone and characterize genomic DNA located upstream of the 3' end ARL2 fragment in this line. Results indicated that this arl2-2 3' end fragment was flanked by a 71-bp DNA sequence of unknown origin (sharing no homologies with sequences present in the databases), itself followed by DNA derived from the 3' end of a putative protein phosphatase 2C gene located on chromosome 2 (At2g20050; Fig. 4B). The recombination breakpoint within At2g20050 was located 537 bp downstream of the A residue of the predicted initiation codon. Both genes were in opposite orientations within the recombined structure (Fig. 4B). To determine if this recombined structure was strictly associated with arl2-2, we tested the ability of At2g20050-specific PCR primers (PP2C-F1 and PP2CR2; Table I; Fig. 4B) to amplify At2g20050 DNA overlapping the recombination breakpoint described in the previous paragraph from wild-type and arl2-2 mutant seedlings. Results shown in Figure 5 indicated that these primers allowed amplification of a 531-bp fragment from wild-type Ws DNA but not from mutant arl2-2 DNA. On the other hand, a 351-bp DNA fragment could be amplified from arl2-2 DNA when a combination of ARL2-specific (ARL2-R7) and At2g20050-specific (PP2C-R2) primers was used in the reaction (Fig. 5). No fragments could be amplified from wild-type DNA with this primer pair (Fig. 5). Taken together, these data suggest that arl2-2 resulted from the combination of a T-DNA insertion within ARL2 and a reciprocal translocation between chromosomes 1 and 2 involving breakpoints within the ARL2 and At2g20050 genes.
Molecular analysis of arl2-3 revealed that this allele derived from insertion of an 8-kb T-DNA within the first predicted intron of the gene. This insertion involved a T-DNA flanked by two left borders and was accompanied by a small 42-bp deletion of predicted intronic sequences 3' of this insertion site (Fig. 4C). We isolated homozygous arl2-2 and arl2-3 plants and tested the gravitropic phenotype of their progeny. Results shown in Figure 1A demonstrated that the roots of arl2-2 and arl2-3 seedlings display altered gravitropism, similarly to arl2-1. Mutant hypocotyls also showed similar alterations in their gravitropic response, whereas mutant inflorescence stems displayed wild-type kinetics of gravitropism (Fig. 2C; data not shown). A cross between homozygous 13-8 (arl2-1) and arl2-2 plants yielded trans-heterozygous progeny that displayed a gravitropic defect in both roots and hypocotyls. Because these mutations were isolated independently in two separate backgrounds (Est and Ws) and both were recessive (data not shown), the data strongly support a role for ARL2 in root and hypocotyl gravitropism. To verify this conclusion, we transformed arl2-1 mutant plants with the p35S-His6::ARL2 construct and analyzed the gravitropic phenotype of the progeny of two independent transformants. Results shown in Figure 1B indicated that seedlings carrying the transgene developed a stronger root curvature response to gravistimulation than untransformed mutant seedlings or than wild-type seedlings of the corresponding ecotype (Est). The gravitropic defect of arl2-1 hypocotyls was also rescued by the p35S-His6::ARL2 construct (data not shown).
ARL2 is one of two genes that encode ARG1-like proteins in Arabidopsis (Fig. 6; Sedbrook et al., 1999
We cloned full-length ARL2 cDNAs (GenBank accession no. AY226826) by RACE-PCR and sequenced them. Three of four cDNA 5' ends were located within a 10-bp A-rich region. The fourth transcription start site was located 53 bp downstream of the first one (data not shown). Two polyadenylation sites were also found at the 3' end, located within 28 bp of each other. The first one was located 136 bp downstream of the translation stop codon (TGA). ARL2 contains 10 exons and nine introns. We also cloned ARL1 cDNA (GenBank accession no. AY226825) and showed that ARL1 contains 10 exons and nine introns, similarly to ARL2. The position of each intron is conserved within these genes (Fig. 4). It is also conserved with the position of introns within ARG1, although the latter gene contains an additional intron within its 3' region (Sedbrook et al., 1999 The first ATG of ARL2 predicted to constitute a good translation initiation site by the Netstart 1.0 program (http://www.cbs.dtu.dk/cgi-bin/) is located 284 bp downstream of the first transcription start site (Fig. 4A). It initiates a large ORF predicted to encode a 415-amino acid polypeptide whose sequence is similar to that of ARG1 and ARL1 throughout their entire lengths (Fig. 6). All three proteins contain a J domain at their N terminus, a potential transmembrane domain, and a putative coiled-coil domain at the C end (Fig. 6). The arl2-1 deletion eliminates all amino acids downstream of R311, deleting most of the coiled-coil domain in the protein. The T-DNA insertion and translocation found in arl2-2 disrupts the protein-coding region at T362, whereas the T-DNA insertion in arl2-3 disrupts the coding potential of ARL2 right at the middle of the J domain (Fig. 6).
The ARG1 gene was shown to be expressed ubiquitously in Arabidopsis, even though the phenotypic analysis of arg1 mutants suggested a specific involvement in gravity signal transduction (Sedbrook et al., 1999
As illustrated in Figures 1, A through D, and 2A, the gravitropic phenotypes associated with mutations in either ARG1 or ARL2 were intermediate between wild-type gravitropism and a complete abolition of the gravitropic response. This afforded an opportunity to genetically test whether the two mutations affected parallel branches of the gravity signal transduction pathway. Homozygous arl2-1 and arg1-2 plants were crossed, and double heterozygous F1 progeny were recovered and shown to develop wild-type responses to gravistimulation (data not shown). arg1-2 arl2-1 double mutants were recovered from the segregating F2 progeny, and the gravitropic phenotype of their selfed progeny was analyzed. Figure 1C shows that arg1-2 arl2-1 double mutants displayed a gravitropic phenotype that was almost identical to the phenotype associated with single mutants. Using similar procedures, we also isolated arl2-1 arl1-4 and arg1-2 arl1-4 double mutants and arl1-4 arl2-1 arg1-2 triple mutants. All double and triple mutants displayed kinetics of root gravitropism that were similar to those of single mutants (Fig. 1C; data not shown).
Because ARG1, ARL2, and PGM all appear to function in gravity perception and/or signal transduction (Kiss et al., 1989
In this paper, we demonstrate that three independently isolated mutations in the ARL2 locus, which encodes a DnaJ-like protein similar to ARG1 (Sedbrook et al., 1999 ), affect root and hypocotyl gravitropism of seedlings grown under light (Fig. 1, B and D) or darkness (Figs. 1, A and C, and 2A). All three mutations are recessive, and do not complement each other. We also show that the gravitropic phenotype associated with arl2-1 can be rescued by transformation with a construct that drives expression of a His-tagged version of the ARL2 protein (Fig. 1B). Together, these data demonstrate that ARL2 is involved in root and hypocotyl gravitropism in Arabidopsis. The agravitropic phenotype associated with arl2-1 and arl2-3 is stronger than that associated with arl2-2 (Fig. 1A). This difference could reflect the relative strengths of these mutations. arl2-1 carries a 3' deletion that eliminates most of a predicted coiled-coil domain at the C terminus of the ARL2 protein, potentially eliminating its ability to interact with target proteins. Similarly, arl2-3 carries a T-DNA insert within its first intron, possibly disrupting the coding potential of this gene within the J domain. These two alleles are likely to be null, although the low level of ARL2 expression in wild-type plants precludes careful verification of this prediction. arl2-2, on the other hand, contains a T-DNA insertion near the 3' end of the gene's ORF, downstream of its coiled-coil-encoding domain (Figs. 4 and 6). Hence, unlike arl2-1 and arl2-3, arl2-2 could potentially encode a functional protein that may interact with its targets.
The arl2-2 mutation involved a complex recombination event that also disrupted a putative protein phosphatase 2C gene (At2g20050). In this line, the 3' end of At2g20050 is associated with the 3' end of arl2-2 and a 71-bp insertion of unknown origin. A second locus carrying the 5' end of ARL2 associated with a T-DNA and the complementary 5' end of At2g20050 was also found in this line (Figs. 4B and 5). No wild-type ARL2 or At2g20050 could be found in this mutant, strongly suggesting that this recombination involved a reciprocal translocation between chromosomes 1 and 2, with breakpoints located at positions 2,550 of ARL2 and 537 of At2g20050 (Fig. 4B). It is not unusual to find chromosomal rearrangements associated with T-DNA insertions in plants (Tax and Vernon, 2001 The hypothesis that mutations in ARL2 are responsible for the gravitropic phenotype found in these mutant lines is supported by the results of transformation experiments in which arl2-1 mutant seedlings were transformed with the p35S-His6::ARL2 construct. The transgenic progeny of independent transformants displayed a strong root gravitropic response relative to untransformed arl2-1 seedlings (Fig. 1B). It is interesting to note, however, that arl2-1 roots overexpressing ARL2 displayed enhanced gravitropism compared with wild-type Est roots (Fig. 1B). This result suggests that ARL2, or a component of the ARL2 pathway, might be limiting for the gravitropic response in the Est background.
Loss-of-function mutations in ARL2 affect root and hypocotyl gravitropism without altering inflorescence stem gravitropism (Figs. 1A and 2, A and C). This result is in agreement with previous observations indicating that the gravitropic responses of different organs are genetically separable (Bullen et al., 1990
It is interesting to note that ARG1, an ARL2 paralog, was also proposed to function in gravity signal transduction, based on a similar combination of phenotypes (Sedbrook et al., 1999
Our analysis of arl2-1 pgm-1 root gravitropism revealed a strong phenotypic enhancement compared with single mutants (Fig. 1D). Similar observations were recently made with arg1-2 pgm-1 double mutants (K. Boonsirichai and P.H. Masson, unpublished data). These striking results strongly suggest that ARL2/ARG1 and PGM function in distinct branches of the gravity signal transduction pathway. Several physiological data support the existence of a secondary gravity perception mechanism, in addition to amyloplast sedimentation within the statocytes (Ishikawa and Evans, 1990
As components of HSP70-containing macromolecular chaperone complexes, DnaJ-like proteins interact with HSP70 through their J domain and with specific targets through a more divergent protein interaction domain (Zuber et al., 1998
Like ARG1, ARL2 is expressed in all tissues of the plant, though at much lower levels (Fig. 7). This result appears to contradict a model postulating a specific involvement of ARL2 in gravity signal transduction. However, as previously discussed for ARG1 (Sedbrook et al., 1999
Finally, we want to emphasize that not all dnaJ-like proteins are needed for root gravitropism. The Arabidopsis genome contains 91 genes encoding DnaJ-like proteins, many more than any other organism whose genome has now been sequenced (Miernyk, 2001
Plant Materials and Growth Conditions To generate a collection of fast neutron-mutagenized seeds, approximately 250,000 seeds of the Est ecotype were exposed to 6,000-Rad fast-neutron radiation by Dr. H. Brunner (Plant Breeding Unit, International Atomic Energy Agency, Vienna). Mutagenized M1 seeds were separated into 29 subpopulations. M2 seeds were harvested in bulk for each subpopulation and examined separately in the gravitropism mutant screen described below.
T-DNA insertional mutants were obtained through the Arabidopsis Knockout Facility (University of Wisconsin Biotechnology Center, Madison) from populations of T-DNA-transformed Arabidopsis plants (Ws ecotype; Krysan et al., 1999
All plant manipulations, including surface sterilization of seeds, plating on agar-based media, growth conditions, transplantation of seedlings from plates to soil, pollination, and harvesting were as previously described (Rutherford and Masson, 1996
Gravitropic Reorientation Assay 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 inflorescence stems. They were positioned horizontally in a dark growth chamber, and the inflorescence stems were photographed at regular time intervals, as described above. Root, hypocotyl, and inflorescence stem curvature responses to gravistimulation were determined on digital images using the National Institutes of Health Image Analysis Software version 1.62 (http://rsb.info.nih.gov/nih-image/).
Root Wave Assay
Hypocotyl phototropism was characterized using the procedures described in Steinitz and Poff (1986
Assays aimed at testing root growth in the presence of phytohormones or polar auxin transport inhibitors were carried out as described by Sedbrook et al. (1999
Data derived from gravitropic and phototropic assays, and root growth responses to added compounds were subjected to statistical analysis (Excel, Microsoft Corporation, Redmond, WA), using Student's t tests to compare mean values between wild-type and mutant populations and F tests to compare deviations from the mean value between genotypes. Differences were assumed to be insignificant when the P values associated with these tests exceeded 0.05.
Seedlings were grown on vertically oriented 0.8% (w/v) agar-based GM medium 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
Two mapping populations were generated by crossing arl2-1 mutant plants with wild-type plants of the Landsberg erecta or Columbia ecotypes, respectively. Their progeny were propagated by self-fertilization. F3 progeny were subjected to a root wave assay to determine their gravitropic phenotype. This progeny typing allowed a determination of the ARL2 genotype of the corresponding F2 parent. Two pools of F3 progeny derived from 15 homozygous wild-type and 15 homozygous mutant F2 plants, respectively, were generated and used to map arl2-1 by bulked segregant analysis (Michelmore et al., 1991
DNA and RNA Isolation
Basic Molecular Biology Procedures
Cloning of ARL2 Genomic and cDNA Sequences The 5' end of the ARL2 mRNA was mapped on the sequenced genomic DNA using the Ambion First-Choice RLM-RACE PCR kit, as recommended by 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, in nested PCR amplifications. PCR-amplified cDNAs were cloned into pBluescript (Stratagene Co., Cedar Creek, TX), and sequenced.
A 3'-RACE procedure was also used to detect ARL2 transcripts in total RNAs extracted from plant organs (Chang et al., 1993
Identification and Characterization of arl2-2 and arl2-3
To investigate the structure of arl2-2, DNA was isolated from homozygous plants, cleaved with NsiI or BglII, and subjected to Southern-blot analysis. Membranes were hybridized successively with PCR-amplified ARL2-specific probes corresponding to sequences flanking the arl2-2 T-DNA on the 5' side, on the 3' side, and to neomycin phosphotransferase II- or
Genomic DNA associated with the arl2-2 3' end segment was isolated by inverse PCR of HindIII-digested genomic DNA after self-ligation (Ochman et al., 1988
To characterize the arl2-3 mutation, arl2-3 genomic DNA was cleaved with NcoI and NdeI and subjected to Southern-blot analysis using ARL2 cDNA and T-DNA left-border probes (Ausubel et al., 1994
Complementation of arl2-1
We introduced the p35S-His6::ARL2 construct in wild-type and mutant plants by in planta transformation (Bent, 2000
Procedures used to clone and characterize the ARL1 cDNA were as described above for ARL2, except that primers ARL1-F1 and ARL1-R1 (Table I) were used in the PCR reactions, along with T3 or T7 primers (Table I). The 5' end of the ARL1 cDNA was mapped on the genomic DNA by RLM-RACE amplification, using the ARL1nestr2 and ARL1nestr1 primers along with Ambion First-Choice RLM-RACE PCR kit primers (Table I; Ambion, Inc). The arl1-4 allele was identified in the BASTA collection of T-DNA insertion lines (Arabidopsis Knockout Facility), using the ARL1-F1 and T-DNA left-border (JL202) primers. Amplified DNA was subjected to nested PCR with JL270 and ARL1nestr2 and sequenced.
ARL1 expression was analyzed by northern-blot analysis of total RNA extracted from seedling and mature plant organs (Fig. 7), using 32P-labeled ARL1 cDNA as a probe (Ausubel et al., 1994
Plants carrying both the arl2-1 and either arg1-2 or pgm-1 mutations at the homozygous state were obtained by crossing single mutants and selfing the corresponding F1 progeny. A cotyledon was excised from each F2 plant and prepared for PCR amplification (Klimyuk et al., 1993 arg1-2 arl2-1 arl1-4 triple mutants were obtained by crossing an arg1-2 arl2-1 double mutant with an arl1-4 single mutant and selfing the corresponding F1 progeny. F2 plants were genotyped as described above for the presence of arg1-2 and arl2-1 at the homozygous state. PCR amplification was also used to identify the arl1-4 allele in DNA extracted from individual cotyledons, using primers ARL1-nestr2 and JL270. Presence of the wild-type ARL1 allele was analyzed by PCR amplification, using the ARL1-F4 and ARL1-nestr2 primers. Individual F2 plants with a specified genotype were grown and selfed. Their progeny were subjected to an in-agar reorientation assay.
We thank the Arabidopsis Knockout Facility (University of Wisconsin, Madison) for providing the arl1-4, arl2-2 and arl2-3 mutants. We also thank Jessica Will and Nicole Ammerman for excellent technical assistance. Received March 11, 2003; returned for revision April 8, 2003; accepted May 22, 2003.
1 This work was supported in part by the National Science Foundation (grant nos. MCB-9905675 and MCB-0240084), by the National Aeronautic and Space Administration (grant nos. NAG2-1336 and NAG2-1602 to P.H.M.), by the National Science Foundation/Department of Energy/U.S. Department of Agriculture (Training Grant fellowship no. BIR 92-2033 to E.S.R.), and by the Thai Government (fellowship to K.B.). This is manuscript no. 3611 from the Laboratory 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 State University, East Lansing, MI 48824. * Corresponding author; e-mail phmasson{at}wisc.edu; fax 608-262-2976.
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-glucuronidase-specific T-DNA probes (Rutherford et al., 1998
