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Keywords:

  • gravitropism;
  • auxin;
  • statocytes;
  • signal transduction;
  • roots

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

ALTERED RESPONSE TO GRAVITY1 (ARG1) and its paralog ARG1-LIKE2 (ARL2) are J-domain proteins that are required for normal root and hypocotyl gravitropism. In this paper, we show that both ARL2 and ARG1 function in a gravity signal transduction pathway with PIN3, an auxin efflux facilitator that is expressed in the statocytes. In gravi-stimulated roots, PIN3 relocalizes to the lower side of statocytes, a process that is thought to, in part, drive the asymmetrical redistribution of auxin toward the lower flank of the root. We show that ARL2 and ARG1 are required for PIN3 relocalization and asymmetrical distribution of auxin upon gravi-stimulation. ARL2 is expressed specifically in the root statocytes, where it localizes to the plasma membrane. Upon ectopic expression, ARL2 is also found at the cell plate of dividing cells during cytokinesis, an area of intense membrane dynamics. Mutations in ARL2 and ARG1 also result in auxin-related expansion of the root cap columella, consistent with a role for ARL2 and ARG1 in regulating auxin flux through the root tip. Together these data suggest that ARL2 and ARG1 functionally link gravity sensation in the statocytes to auxin redistribution through the root cap.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Gravitropism allows plant organs to orient their growth within the gravity field. Changes in orientation relative to gravity (gravi-stimulation) are met by corrective bending toward the original growth direction. The gravitropic response of primary roots has been conceptually separated into four phases: gravity susception, signal transduction, signal transmission, and differential growth (Blancaflor and Masson, 2003). Gravity sensing occurs in large part within specialized cells called statocytes that are located in the cap of roots and the endodermis of hypocotyls and shoots. Statocytes contain dense, starch-filled amyloplasts (statoliths), whose position or movement within the cell provide information about the organ’s orientation relative to the gravity vector. There is abundant evidence that amyloplasts are involved in gravity perception. However, to date there has been no definitive description of the mechanism that senses the position or movement of amyloplasts within the statocytes (Perrin et al., 2005). It has been proposed that sedimenting amyloplasts may trigger mechano-sensitive ion channels at the plasma membrane or ER, either directly, by sedimentation onto them (Sack, 1991; Zheng and Staehelin, 2001), or indirectly, by exerting pressure on the actin cytoskeleton (Sievers et al., 1989; Volkmann and Baluska, 1999) or disrupting its meshwork upon sedimentation (Yoder et al., 2001). The latter model may have to be reconsidered in light of studies indicating that low concentrations of the actin-depolymerizing drug latrunculin B enhance gravitropism rather than inhibiting it (Hou et al., 2003, 2004; Yamamoto and Kiss, 2002). Alternatively, the ligand–receptor model developed to explain gravity perception in Characean rhizoids postulates that statolith-borne ligands interact physically with receptors carried by sensitive membranes along the sides of statocytes, thereby triggering gravity signal transduction in these cells (Limbach et al., 2005). Finally, it is also possible that pressure exerted by the protoplast upon the surrounding cell wall might act as a gravity sensor (Staves et al., 1997).

Following gravity perception, signal transduction leads to lateral polarization of the statocytes, which is thought to direct the passage of information toward the elongating cells where differential growth occurs (Blancaflor and Masson, 2003). Several events have been implicated in the signal transduction phase of root gravitropism, such as cytoplasmic alkalinization in root cap statocytes, accompanied by acidification of the apoplast and polar accumulation of the auxin efflux facilitator PIN3 at the new lower side of the statocytes (Fasano et al., 2001; Friml et al., 2002; Scott and Allen, 1999; Young et al., 2006). Asymmetrical accumulation of PIN3 in the columella cells is thought to drive lateral auxin redistribution toward the new lower side of the root (Friml et al., 2002).

There is abundant evidence that lateral redistribution of auxin is important for the signal transmission and growth response phases of gravitropism. Lateral redistribution of auxin in roots can be detected by following the movement of radio-labeled auxin (Young et al., 1990), or visualizing the expression of reporter genes driven by the auxin-responsive DR5 promoter (Boonsirichai et al., 2003; Ottenschläger et al., 2003; Rashotte et al., 2001). Additionally, the auxin-dependent production of reactive oxygen species, as monitored by dihydro-rhodamine-123 fluorescence, can be detected asymmetrically along the lower flank of gravi-stimulated corn roots (Joo et al., 2001). The development of auxin asymmetry is thought to derive from redirection of auxin through the root tip toward the new lower flank of the root cap, followed by basipetal transport to the elongation zone (Blancaflor and Masson, 2003; Swarup et al., 2005). Basipetal transport of auxin from the cap to the elongation zone requires both auxin influx and efflux carriers, encoded by AUX1 and AGR1/PIN2/EIR1/WAV6, respectively (reviewed in Chen et al., 1999; Swarup et al., 2005), and members of the MDR/PGP ABC transporter families (Geisler and Murphy, 2006; Lewis et al., 2007; Lin and Wang, 2005; Terasaka et al., 2005). Basipetal auxin transport may also be regulated by molecules affecting the activity, expression and subcellular localization of auxin transporters in the lateral cell files of the root (as reviewed by Muday and Rahman, 2007). In addition to auxin, other hormones or signaling molecules such as cytokinins (Aloni et al., 2004), reactive oxygen species (Joo et al., 2001), flavonoids and ethylene (Buer and Muday, 2004; Buer et al., 2006) may be involved in the growth response phase of gravitropism through control of differential elongation, either in parallel with auxin or as regulators of the auxin-mediated signaling pathway.

Mutations in two paralogous Arabidopsis genes, ARG1/RHG1 and ARL2, result in slower kinetics of root and hypocotyl gravitropism (Fukaki et al., 1997; Guan et al., 2003; Sedbrook et al., 1999). arg1 and arl2 mutants respond normally to auxin and auxin transport inhibitors and show normal phototropic bending, indicating that ARG1 and ARL2 function specifically in gravity sensing/signaling (Guan et al., 2003; Sedbrook et al., 1999). The arg1-2 mutant lacks alkalinization of the columella cytoplasm and redistribution of auxin across the root tip following gravi-stimulation (Boonsirichai et al., 2003). arg1 and arl2 single mutants as well as arg1 arl2 double mutants show similar gravitropic defects, indicating that ARG1 and ARL2 function in the same gravity-signaling pathway (Guan et al., 2003). ARG1 and ARL2 each contain a J-domain near their N-termini, and a predicted coiled-coil motif near their C-termini (Guan et al., 2003; Sedbrook et al., 1999). J-domain proteins are present in organisms of all kingdoms, and make up a diverse gene family in Arabidopsis (Miernyk, 2001). They typically function in the molecular chaperone system by physically interacting with heat shock cognate 70 chaperones, modulating their ATPase and substrate binding activities (Mayer and Bukau, 2005). Through these interactions, they mediate changes in the folding, activity, targeting, abundance and/or interactions of a variety of protein substrates.

ARG1 is a peripheral membrane protein associated with several cellular membranes of the endomembrane system, including membranes at the cell plate and the plasma membrane (Boonsirichai et al., 2003). Additionally, a small amount of ARG1 protein may associate with actin, an interaction that has been predicted from its primary structure (Boonsirichai et al., 2003; Sedbrook et al., 1999). It has been postulated that ARG1 functions in gravitropism by mediating changes in the activity and/or localization of proteins that are directly involved in gravity sensation and/or signal transduction (Boonsirichai et al., 2003). Candidates for these substrate proteins include PIN3 and other proteins that may mediate auxin transport through the root tip (Friml et al., 2002; Perrin et al., 2005), as well as proteins that may contribute to cytoplasmic alkalinization, such as H+-pyrophosphatases and members of the multi-subunit H+ ATPase family (Li et al., 2005).

In this paper, we show that both ARL2 and ARG1 are required for lateral redistribution of auxin upon gravi-stimulation and for the accumulation of PIN3 at the new lower side of root statocytes. Additionally, we demonstrate that mutations in ARL2 and ARG1 eliminate the contribution of PIN3  to the root gravitropic response. We show that ARL2 is specifically expressed in root statocytes, and report on its subcellular localization at the cell plate and plasma membrane. Finally, we report that ARL2 and ARG1 regulate root cap morphology, another auxin-regulated process. Together, these data support a model whereby ARL2 and ARG1 function in a gravity-signaling complex in statocytes.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

ARL2 and ARG1 are required for lateral redistribution of auxin-responsive reporter expression across a gravi-stimulated root tip

To analyze the function of ARL2 in regulating asymmetrical redistribution of auxin, we analyzed the expression of transgenic reporters that fuse the auxin-responsive DR5 promoter to either β-glucuronidase (DR5::GUS) or GFP (DR5rev::GFP) in arl2-3, arg1-2 and wild-type root tips both before and after gravi-stimulation (Friml et al., 2003; Ulmasov et al., 1997). After 6 h of gravi-stimulation, lateral activation of GUS expression on the lower side of the root tip was detected in wild-type controls but was absent in all arl2-3 and arg1-2 root tips analyzed (Figure 1a). Analysis of DR5rev::GFP expression in live seedlings was performed by growing seedlings expressing DR5rev::GFP in agar against a cover slip mounted on a vertically oriented microscope with a rotating stage (see Experimental procedures). This allowed visualization of the same root throughout the gravitropic response, and was important because asymmetric expression of both DR5::GUS and DR5rev::GFP reporters also occurs in vertically oriented roots, making it difficult to attribute asymmetrical GUS activity to gravity-regulated redistribution (Table 1) (Young et al., 2006). By comparing signals in roots just after gravi-stimulation (time 0) to those at later time points, we were able to identify post-stimulation expression patterns. Under these conditions, dramatically asymmetric expression of DR5rev::GFP was seen 8 h after stimulation within the lateral cap and epidermal cells of the distal elongation zone. During these experiments, we observed instances of asymmetrical expression along the upper flank of some gravi-stimulated root tips. To determine the gravity-regulated proportion of asymmetrical signals, we subtracted the number of roots showing signal along the upper flank from those showing signal along the lower flank and divided this value by the total number of roots observed (Table 1).

image

Figure 1.  Asymmetrical expression of DR5 reporters is attenuated in arl2-3 and arg1-2 mutants. DR5 reporter expression in roots grown either on (a) or within (b) nutrient agar. (a) Representative images of DR5::GUS expression in roots after 6 h of gravi-stimulation. Arrows indicate direction of gravity. Notice the asymmetrical GUS expression on the lower side of wild-type (Ws) root caps and not in arg1-2 and arl2-3. (b) Representative images of DR5rev::GFP in roots during the gravitropic response. The duration of gravi-stimulation is shown; gravity vector is down in all frames. Note the asymmetrical GFP signal along the lower flank of WT roots (arrowheads) and the symmetrical signal in the same regions of arg1-2 and arl2-3 roots. Scale bars = 20 μm (a) and 50 μm (b). The images are representative of at least 18 roots per genotype (see Table 1).

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Table 1. ARG1 and ARL2 are required for asymmetrical redistribution of DR5rev::GFP expression following gravi-stimulation
Time (h)Wild-typearl2-3/arl2-3arg1-2/arg1-2
  1. Percentages were calculated as 100 × (number of roots showing asymmetrical expression on the lower flank − number of roots showing asymmetrical expression along the upper flank)/total number of roots. The total numbers of roots analyzed per genotype per time point are shown in parentheses. Values for time zero reflect the number of vertically grown roots showing asymmetrical expression in either lateral direction.

 030% (40)31% (41)17% (41)
 871% (24)6% (18)8% (25)
1237.5% (8)0% (8)14% (7)
17−14% (7)−10% (10)0% (8)

For wild-type roots, 71% showed asymmetrical expression of DR5rev::GFP on the lower flank following 8 h of gravi-stimulation (Figure 1b and Table 1). This proportion decreased to 37.5% after 12 h of stimulation. After 17 h, wild-type roots occasionally displayed an opposite gradient in DR5rev::GFP expression, with preferential expression on the upper flank of the root (Table 1). In comparison, arl2-3 [DR5rev::GFP] and arg1-2 [DR5rev::GFP] roots showed greatly attenuated responses after 8 h, with only 8% and 6% of roots, respectively, displaying asymmetrical expression on the lower flank (Figure 1b and Table 1). The extent of asymmetrical DR5rev::GFP expression in arl2-3 and arg1-2 was also less dramatic than that in the wild-type. Similarly, at 12 and 17 h after gravi-stimulation, expression of DR5rev::GFP in arl2-3 and arg1-2 was only rarely asymmetrical, indicating that auxin redistribution is not simply delayed in either mutant (Table 1).

ARL2 and ARG1 function in the same genetic pathway as PIN3 in root gravitropism

ARL2 and ARG1 encode similar J-domain proteins and may function in gravitropism by modulating the activity, localization or complex formation of other target proteins (Perrin et al., 2005). One candidate substrate for ARL2 and ARG1 in gravitropism is PIN3. If PIN3 activity/localization depends on ARL2 and ARG1, arl2 pin3 and arg1 pin3 double mutants will not display a gravitropic phenotype that is more dramatic than that of the single mutants. If, however, PIN3 functions in a gravitropic response pathway that is ARL2- and ARG1-independent, the gravitropic response of arl2 pin3 and arg1 pin3 double mutants will be more severely affected than that of either single mutant.

In order to perform double mutant analysis in the same genetic background, we isolated Columbia (Col-0) alleles of ARL2 (arl2-4) and ARG1 (arg1-3) and crossed them to the null pin3-3 Col-0 allele (Friml et al., 2002). arl2-4 harbors T-DNA in the 2nd exon of ARL2, 327 nt from the start codon, whereas arg1-3 carries T-DNA within the 9th intron of ARG1, 1971 nt from the start codon (Figure S1). Analysis of transcription from arl2-4 indicated that this allele produces a transcript that results from splicing together the 1st and 3rd exons, thereby excising the 2nd exon and T-DNA (Figure S1). Sequencing of the corresponding cDNA segment revealed that the 1st and 3rd exons of ARL2 are out of phase, so the mutant mRNA encodes only 57 amino acids followed by a stop codon within the highly conserved J-domain. Analysis of transcription from arg1-3 by RT-PCR indicated that no full-length transcript is produced from this allele (Figure S1). Both arl2-4 and arg1-3 are therefore likely to be null alleles. Additionally, arl2-4 and arg1-3 failed to complement arl2-3 and arg1-2 mutations, respectively, indicating that the gravitropic phenotypes of arl2-4 and arg1-3 are due to mutations in ARL2 and ARG1 (Figure S1).

Roots of arl2-4 pin3-3 and arg1-3 pin3-3 double mutants showed gravitropic kinetics that were indistinguishable from those of arl2-4 and arg1-3 single mutants, respectively (Figure 2a,b). These results indicate that PIN3 functions in the ARL2 and ARG1 pathway to mediate root gravitropism.

image

Figure 2. PIN3 functions in the ARL2–ARG1 pathway in root, but not hypocotyl, gravitropism. Root (a, b) and hypocotyl (c, d) gravitropism of wild-type (Col), single arl2-4 and pin3-3, and double arl2-4 pin3-3 mutant seedlings (a, c), and of wild-type, single arg1-3 and pin3-3, and double arg1-3 pin3-3 mutant seedlings (b,d). Data are means ± SE for = 88–176 (a), 101–169 (b), 45–56 (c) and 71–90 (d), from two replicates. Some error bars are hidden by the symbols. Significant differences (< 0.05, t test) are labeled with one asterisk where reference is to the wild-type, or two asterisks where reference is to the most severe single mutant. Each assay was repeated once with similar results.

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Surprisingly, the hypocotyls of arl2-4 pin3-3 and arg1-3 pin3-3 seedlings showed slower gravitropic bending at later time points than either arl2-4 or arg1-3 single mutants respectively (Figure 2c,d). These results demonstrate that ARL2 and ARG1 function in a distinct pathway from PIN3 to mediate hypocotyl gravitropism. However, the mean angles for double arl2-4 pin3-3 and arg1-3 pin3-3 mutants at the early time points of the hypocotyl gravitropic response (3 and 6 h) are similar to the arl2-4 and arg1-3 single mutant angles.

Both ARL2 and ARG1 are required for the accumulation of PIN3 on the new lower side of columella cells in gravi-stimulated roots

Upon gravi-stimulation, PIN3 accumulates on the new lower side of root statocytes and is thought to drive lateral redistribution of auxin to the lower flank of the root (Friml et al., 2002; Young et al., 2006). Mutations in ARL2 and ARG1 attenuate auxin redistribution (Figure 1 and Table 1) (Boonsirichai et al., 2003), and eliminate the contribution of PIN3 to root gravitropism (Figure 2). We therefore hypothesized that arl2 and arg1 mutants have altered expression or localization of PIN3. In order to determine the effect of mutations in ARL2 and ARG1 on the expression or subcellular localization of PIN3 in columella cells, we immunolocalized PIN3 in arl2-3, arg1-2, and Wassilewskija (Ws, wild-type) roots before and after gravi-stimulation, using anti-PIN3 antibodies (Young et al., 2006). Root tips were cut and collected into fixative either from vertically oriented seedlings (vertical samples) or from seedlings gravi-stimulated by rotating the plates 90° (stimulated samples). Stimulated samples were cut diagonally across the gravity vector such that their orientation within the gravity field could be determined unambiguously after staining.

PIN3 was symmetrically localized at the cell periphery and within the cytoplasm in columella cells of vertically grown arl2-3, arg1-2 and Ws roots (Figure 3). This result indicates that there is no apparent defect in expression or peripheral localization of PIN3 within statocytes of vertically growing arl2-3 and arg1-2 roots. Upon gravi-stimulation, PIN3 was asymmetrically localized to the new lower side of the columella cells of about 50% of wild-type root tips after 10, 20 or 40 min of stimulation (Figure 3 and Table 2). Consistent with the results of Young et al. (2006), we did not see asymmetrical accumulation of PIN3 signals in all stimulated wild-type root tips or in every cell of any root tip. Importantly, accumulation toward the new topside was observed in only one wild-type sample, with the rest showing no detectable bias (Table 2). These results demonstrate a clear bias toward lateral accumulation of PIN3 on the new lower side of columella cells of gravi-stimulated wild-type root tips, as proposed by Friml et al. (2002) and reported by Young et al. (2006). PIN3 did not show lateral accumulation to the new lower side of statocytes in either arl2-3 or arg1-2 (Figure 3 and Table 2). The lack of asymmetrical PIN3 accumulation in arl2-3 and arg1-2 persisted in roots stimulated for 40 min, indicating the absence or severe attenuation of this response to gravi-stimulation in both mutants.

image

Figure 3.  PIN3 does not accumulate at the new lower side of root statocytes in gravi-stimulated arl2-3 and arg1-2 mutants. Representative confocal sections through the central root caps of vertical or gravi-stimulated roots. After treatment, roots were fixed and immunostained with anti-PIN3 antibodies. Arrows indicate the gravity vector. The duration of gravi-stimulation is shown. Note the accumulation of PIN3 on the lower side of tier 2 and tier 3 columella cells in wild-type (Ws) root tips after 20 and 40 min gravi-stimulation (red arrowheads). This polar localization was absent in arg1-2 and arl2-3. Scale bar = 20 μm. Images are representative of at least seven samples per genotype (see Table 2).

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Table 2. ARL2 and ARG1 are required for PIN3 relocalization in root cap columella cells
GenotypeDuration of stimulus (min) Lowera Upperb No biascTotal RIPIN3d
  1. aNumber of root tips showing polar PIN3 localization on the new lower side of the columella cells.

  2. bNumber of root tips showing polar PIN3 localization on the new upper side of the columella cells.

  3. cNumber of root tips where PIN3 localization showed no bias toward lower or upper sides of the columella cells.

  4. dRIPIN3, relocalization index for PIN3 = 100 × (number of roots showing bias toward the lower side − number showing bias toward the upper side)/total number of roots.

Ws10504956
arg1-21011128
arl2-300220
Ws205151136
arg1-21012138
arl2-31115170
Ws40404850
arg1-20167−14
arl2-300770

ARL2pro::ARL2-GFP is specifically expressed in root statocytes, and ARL2–GFP localizes to the plasma membrane and cell plate

ARG1 is expressed throughout the plant, and localizes to several organelles and membranes including the plasma membrane (Boonsirichai et al., 2003). In order to determine the expression pattern and subcellular localization of ARL2, we created two constructs, fusing either the ARL2 genomic region, including the promoter, to GFP (ARL2pro::ARL2-GFP), or fusing the ARL2 cDNA, controlled by the strong, constitutive 35S promoter, to GFP (35S::ARL2-GFP), and analyzed their expression in transformed Arabidopsis by confocal microscopy (Figures 4 and 5). When expressed from its own promoter, ARL2–GFP shows a striking expression pattern, restricted to tiers 1, 2 and 3 of the root cap columella (Figure 4a,d). Closer inspection of ARL2–GFP in these cells revealed that most of the signal is associated with the cell periphery, with weaker cytoplasmic signal occasionally observed in cells with high expression levels (Figure 4e). Under the control of the 35S promoter, ARL2–GFP localizes to the periphery of all cells in which it can be detected. In root epidermal, cortical and lateral cap cells, it co-localizes with AGR1/PIN2 in membranes that carry the latter protein (Figure 5a). Interestingly, ARL2–GFP signals were most intense at the newly forming cell wall (cell plate) in dividing cells (Figure 5a,b). Note that at least five independent transformants for each construct showed similar signals, and both transgenic ARL2–GFP fusions rescue the gravitropic defect of arl2-3 (Figure 4f, and data not shown), indicating that the fusion protein is functional. Plants expressing either 35S::ARL2-GFP or ARL2pro::ARL2-GFP showed only weak fluorescence in hypocotyls (not shown), suggesting that ARL2 might be subject to post-transcriptional regulation. ARL2–GFP also co-fractionated with membranes upon centrifugation, indicating that ARL2 is membrane-associated (Figure 5c). Additionally, ARL2–GFP signals remained within the protoplasm upon brief plasmolysis, indicating that ARL2 is not associated with the cell wall (Figure 5d). Together, these results indicate that ARL2 is expressed in root statocytes and associates primarily with the plasma membrane.

image

Figure 4.  ARL2 is expressed specifically in root statocytes. Confocal micrographs of GFP fluorescence (green) and propidium iodide counter-stain (purple) in root tips of 4–day-old arl2-3 [ARL2pro::ARL2-GFP] (a, c) and wild-type (Ws) control (b, d). (e) Multi-photon micrograph of GFP fluorescence in columella cells of arl2-3 [ARL2pro::ARL2-GFP], showing peripheral distribution in the statocytes (inset shows WT control columella under similar microscopy settings). Scale bars = 50 μm (a, b), 20 μm (c, d), and 10 μm (e). (f) Root gravitropism of two independent arl2-3 lines expressing ARL2pro::ARL2-GFP, compared to arl2-3 and wild-type. Data are means ± SE of 49–96 individuals per genotype from two replicates. Some error bars are hidden by symbols. Significant differences (P < 0.05, t test) from the wild-type are marked by an asterisk.

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image

Figure 5.  ARL2 localizes to the plasma membrane and cell plate. (a) Co-localization of ARL2–GFP with PIN2 at the plasma membrane and cell plate (arrowhead) in epidermal cells of the distal elongation zone. The inset is a non-transgenic control. (b) Localization of ARL2–GFP at the cell plate (arrowhead) defined by tubulin in the surrounding phragmoplast of a dividing lateral cap cell. Images in (a) and (b) are of fixed arl2-3 seedlings expressing 35S::ARL2-GFP and immunostained with anti-AGR1/PIN2 (a) or anti-α-tubulin (b). (c) Fractionation of ARL2–GFP, PEP12 (an integral membrane protein control) and PUX1 (cytosolic protein control) in the total protein extract (S1), and in the soluble (S150) and microsomal membrane fractions (P150). The results are representative of two experiments. (d) ARL2–GFP in epidermal cells of the distal elongation zone of roots treated with water or plasmolyzed with 0.8 m mannitol for 10 min, and stained with either propidium iodide (PI) to show cell walls or FM4-64 to show membranes. The results shown are representative of at least nine roots per treatment. Similar results were obtained using 1 m KNO3 for plasmolysis. Images are of 3–4-day-old seedlings. Scale bars = 10 μm (a), 5 μm (b, d).

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Mutations in ARL2 and ARG1 affect the number of columella cell files in the root cap

Interestingly, PIN3 protein was found in an expanded number of root tip cells in arl2-3 and arg1-2 compared to Ws (Figure 3), as were DR5rev::GUS (Figure 6a) (Boonsirichai et al., 2003) and DR5rev::GFP expression (Figure 6b). These results suggested that there may be more cells with columella identity in arl2-3 and arg1-2 root tips. To investigate this possibility, we stained root tips for starch to identify columella cells. We observed an increase in the number of columns of starch-staining cells in arl2-3 (5.8 ± 0.46, mean ± SD, P value < 2 × 10−31, t test) and arg1-2 (5.9 ± 0.60, P value < 4 × 10−26) compared to wild-type (4.3 ± 0.60, Figure 6c). We also noted the occurrence of arl2-3 and arg1-2 root caps with additional cell files shaped like central columella cells (Figure 6d). These results indicate that there are more files of cells with columella cell identity in mutant root tips compared to wild-type, but the number of tiers is unaffected.

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Figure 6. arl2-3 and arg1-2 affect the numbers of root cap columella columns. Expression of DR5::GUS (a) and DR5rev::GFP (b) in root caps of 4-day-old vertically oriented seedlings. (c) Lugol-stained root tips showing starch. (d) Propidium iodide staining in root tips showing additional columella-like cell files (asterisks) in arg1-2 and arl2-3 root caps. The wild-type is in the Ws background. Scale bars = 20 μm. Data are representative of at least eight seedlings per genotype.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Mutations in ARL2 or ARG1 dramatically attenuate lateral auxin redistribution to the lower flank of gravi-stimulated roots, as visualized by the expression of DR5-based reporters (Figure 1 and Table 1) (Boonsirichai et al., 2003), eliminate the contribution of PIN3 to the root gravitropic response (Figure 2), and fail to accumulate PIN3 on the new lower side of statocytes (Figure 3). These results indicate that ARL2 and ARG1 are required for PIN3 localization and/or function, and for the lateral redistribution of auxin through the root cap upon gravi-stimulation.

That ARL2 and ARG1 are needed for full lateral redistribution of auxin across the root cap upon gravi-stimulation was documented by determining the expression of the auxin-responsive DR5::GUS or DR5rev::GFP reporters in the root tip of control and gravi-stimulated roots (Figure 1 and Table 1). In these experiments, a large proportion of wild-type roots displayed asymmetrical DR5::reporter expression at their lower flank within 8 or 12 h of gravi-stimulation, whereas arl2-3 and arg1-2 roots did not (Table 1). Interestingly, a small fraction of wild-type roots displayed an opposite asymmetrical expression of these reporters across the tip, with enhanced expression along the upper flank, when tested after 17 h of gravi-stimulation (Table 1). Such roots may be in the process of developing an opposite, counter-curvature at the end of their gravi-response, as previously documented (Ishikawa et al., 1991; Zieschang and Sievers, 1991).

Expression of ARL2–GFP under its own promoter is specifically enriched in the S2 tier of the root cap columella (Figure 4). These cells are probably the primary site of gravity sensation and signal transduction in roots. Indeed, this is where amyloplast sedimentation is the fastest, laser ablation of these cells results in the most dramatic reduction in gravity sensitivity (Blancaflor et al., 1998), PIN3 is expressed (Friml et al., 2002), and gravity-dependent cytoplasmic pH change is the most dramatic (Fasano et al., 2001; Scott and Allen, 1999). Furthermore, expression of ARL2–GFP in these cells (by the ARL2 promoter) is sufficient to rescue arl2-3 root gravitropism (Figure 4e). Statocyte-specific expression of ARG1 is also sufficient to rescue arg1-2 gravitropism (Boonsirichai et al., 2003). Together, these data demonstrate that ARL2 and ARG1 function in the root statocytes to modulate root gravitropism, linking reorientation to lateral polarization of statocytes and auxin redistribution.

The mechanism by which PIN3 accumulates laterally following gravi-stimulation is not known. Although differential protein turnover has not been excluded, alteration in vesicle-mediated PIN3 trafficking is probably involved. Gravi-stimulation could re-target vesicles containing PIN3 to the new lower side, or shift the balance of PIN3 cycling toward plasma membrane deposition along the lower side and/or toward removal from the plasma membrane along the upper side. Factors affecting this response may act on PIN3 localization by regulating vesicle trafficking. Alternatively, alterations in PIN efflux activity may themselves affect PIN localization at the plasma membrane (Paciorek et al., 2005). ARG1 is localized throughout the vesicle trafficking pathway, and both ARL2 and ARG1 localize to the plasma membrane and accumulate at the cell plate when expressed in dividing cells (Figure 5) (Boonsirichai et al., 2003). During cytokinesis, much of the secretory and vesicle trafficking activity is directed toward the cell plate, where new cell-wall materials, membrane and membrane proteins are being deposited (Jürgens, 2005). The prominent localization of ARG1 and ectopically expressed ARL2 to the cell plate suggests that they are present in areas of high vesicle trafficking activity. Mutations in ARL2 or ARG1 do not affect the localization or expression of PIN2 (Figure S2), and the mutants show normal growth responses to the auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA) and unilateral light (Guan et al., 2003; Sedbrook et al., 1999). Therefore, if ARL2 and ARG1 function in PIN3 trafficking, they do so specifically within the gravity-signaling pathway. ARL2 and ARG1 may function in gravity signaling to link gravity sensation to changes in vesicle dynamics and/or the activity of membrane-associated proteins and lateral auxin transport. Interestingly, ARL2–GFP appeared to dissociate from the plasma membrane upon brief plasmolysis (≤ 10 min treatment with 0.8 m mannitol, Figure 5d). Hydrotropism and water stress in Arabidopsis and Raphanus sativus roots have been shown to inhibit gravitropism through amyloplast degradation (Takahashi et al., 2003). It may be that osmotic stress also stimulates membrane dissociation of ARL2, thereby altering gravity signaling. More work is underway to address this exciting possibility.

Interestingly, PIN3 shows accumulation at the new lower side of statocytes in only about 50% of wild-type roots at any of the time points analyzed here. PIN3 has been hypothesized to function in redirection of auxin toward the new lower flank of gravi-stimulated roots (Friml et al., 2002). It may be that the modest level of PIN3 accumulation detected in this study is sufficient to redirect auxin asymmetrically through the cap, or that PIN3 is only a minor player in lateral redirection of auxin upon gravi-stimulation. The weak root gravitropic phenotype of two null alleles of PIN3 (pin3-3 and pin3-4), and the evidence for functional redundancy among the PIN gene family, suggests that auxin redistribution in the root cap is mediated by proteins in addition to PIN3, perhaps more so in pin3 mutants (Blilou et al., 2005; Vieten et al., 2005). It remains to be seen whether ARL2 and ARG1 also function in these redundant pathways. Lateral accumulation of PIN3 may also be transient, undergoing dynamic association and dissociation with the new lower membrane, and therefore may not be detectable in all cells or in all roots at any given time. This becomes more obvious after 40 min of gravi-stimulation, when fewer columella cells per root displayed asymmetrical PIN3 localization at the plasma membrane (Figure 3). Additionally, PIN3 relocalization may be sensitive to stimuli other than gravity, such as touch or light. Indeed, small and transient touch stimulation inhibits gravi-sensitivity and reduces amyloplast sedimentation velocity in roots (Massa and Gilroy, 2003). Hence, touch stimulation could also affect PIN3 relocalization. The experimental procedures employed here were designed to minimize stimuli other than reorientation within the gravity field. However, stimuli associated with root tip collection and fixation could not be eliminated. Alternative means of visualizing PIN3 relocalization, such as the use of live reporters, may help resolve these issues.

We also note that the pin3-3 root gravitropism phenotype is weak in our experiments compared with that described by Friml et al. (2002). However, the experiments performed here using pin3-3, arl2-4 pin3-3, and arg1-3 pin3-3 were repeated with similar results, and another allele of PIN3 (pin3-4) also showed similar phenotypes to pin3-3 in our hands (data not shown). Hence, the differential gravitropic phenotype associated with the same pin3 mutants in different laboratories may reflect differences in experimental conditions.

Roles for ARL2 and ARG1 in regulating auxin flux through the root cap are also supported by the apparent accumulation of auxin in arl2-3 and arg1-2 root tips, as indicated by increased expression of the DR5::GUS and DR5rev::GFP reporters, and increased number of columella cell files as defined by starch staining and PIN3 expression (Figure 6) (Boonsirichai et al., 2003). Indeed, auxin has been implicated in regulating the extent of meristem activity in the root, whereby high auxin levels in the root cap result in differentiation of more cells with columella identity, and the converse for roots with reduced auxin levels in the cap (Sabatini et al., 1999). Accordingly, when we analyzed the number of cell files expressing DR5rev::GFP and displaying starch staining in the same roots for arl2-3, arg1-2 and the wild-type, we noticed a correlation between the two parameters, with a coordinate increase in both measurements in arl2-3 and arg1-2 mutants (Figure S3).

The hypocotyl gravitropic response of double arl2-4 pin3-3 and arg1-3 pin3-3 mutants at early time points (3 and 6 h) is similar to the arl2-4 and arg1-3 single mutant response (Figure 3c,d). However, hypocotyl gravitropism of both double mutants at later time points was enhanced compared to the single mutants (Figure 3c,d). These data indicate that PIN3 may function in the ARL2 and ARG1 pathway during the early hypocotyl gravitropic response, and in an ARL2- and ARG1-independent pathway in the later phases of hypocotyl gravitropism. These results are interesting considering that the endodermis is the gravity-sensing site in the hypocotyl (Morita and Tasaka, 2004), where PIN3 is expressed (Friml et al., 2002) and where ARG1 expression is sufficient to rescue the arg1-2 hypocotyl gravitropism defect (Boonsirichai et al., 2003). The results suggest that PIN3, ARL2 and ARG1 function in distinct gravitropic pathways in the same cells. Neither arg1 nor arl2 mutations affect phototropic bending in hypocotyls (Guan et al., 2003; Sedbrook et al., 1999), while pin3 affects both hypocotyl gravitropism and phototropism (Friml et al., 2002). Together, these data suggest that ARL2 and ARG1 are not general regulators of PIN3 function; both function more specifically in early gravity signaling, possibly by modulating the trafficking of PIN3 protein to and from the plasma membrane of the statocytes, as discussed above for roots. These results also further document the differences in gravity signal transduction between hypocotyls and roots (Morita and Tasaka, 2004), and suggest that the same molecules may function in both responses in different ways. Further studies will be aimed at elucidating the functional and molecular relationships linking ARL2, ARG1 and PIN3 during root and hypocotyl gravitropism.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials and growth conditions

arl2-4 (SALK_034413), arg1-3 (SALK_024542) and pin3-4 (SALK_038609) were isolated by screening the SALK T-DNA insertion collection, in the Col background (Alonso et al., 2003). arg1-2 and arl2-3 mutants, in the Ws background, have been described previously (Guan et al., 2003; Sedbrook et al., 1999); these alleles are probably null. DR5::GUS-transformed seeds in the Col background (Ulmasov et al., 1997) were obtained from T.J. Guilfoyle (University of Missouri, Columbia, MO). Seeds of the pin3-3 allele (Friml et al., 2002), as well as DR5rev::GFP-transformed seed in the Col background (Friml et al., 2003), were obtained from J. Friml (University of Tübingen, Germany). For analysis of DR5::GUS and DR5rev::GFP expression, arg1-2 and arl2-3 were crossed to the DR5::GUS- and DR5rev::GFP-transformed lines. Seed for analysis was collected from F3 families that were either homozygous for arg1-2 or arl2-3, or homozygous for ARG1 and ARL2 (wild-type controls) and non-segregating for GUS activity or GFP fluorescence. arg1-3 pin3-3 and arl2-4 pin3-3 double mutants were isolated from F4 populations. All plant manipulations, including surface sterilization of seeds, plating on agar-based medium, and growth conditions were as previously described (Rutherford and Masson, 1996).

Vertical stage microscopy

For analysis of DR5rev::GFP expression, seed was sterilized and cold-treated for 3 days, then sown on approximately 1 ml of half-strength Linsmeier and Skoog (LS) medium containing 0.5% sucrose and 0.8% agar that had been solidified on sterile cover slips. Cover slips were then sealed in petri plates with parafilm, tilted back 30° from the vertical and placed into a growth chamber such that the roots grew along the cover slip surface. After 4–5 days of growth, cover slips were removed and sealed in plexiglass slides. Slides were mounted on a vertical-stage microscope (Nikon Optiphot-2; http://www.nikon.com, tilted on its back) equipped with a standard FITC/GFP filter cube. Seedlings were allowed to acclimatize for > 30 min before each experiment. Gravi-stimulation was performed by rotating the microscope stage 90°. Digital images were taken using a CCD camera (Diagnostic Instruments; http://www.diaginc.com). For analysis of DR5rev::GFP expression and starch staining in the same root, seedlings were mounted on slides and images of GFP fluorescence were collected prior to lugol staining for starch visualization.

Tropism assays

Root and hypocotyl gravitropism experiments were carried out as described previously (Boonsirichai et al., 2003), using 3–5-day-old seedlings, which, in our hands, develop more uniform gravi-responses than older seedlings. For hypocotyl gravitropism, seed was plated on nutrient medium containing 2% agar, stratified, treated with light for 24 h to induce germination, then wrapped in aluminum foil for 3 days of etiolated growth. Gravi-stimulation was provided by 90° rotation of the plates. Hypocotyl angles were measured from digital images using NIH Image, version 1.63 (http://rsb.info.nih.gov/nih-image/).

Molecular cloning and plant transformation

For the 35S::ARL2-GFP construct, the ARL2 cDNA was PCR-amplified without its stop codon from a Col-0 cDNA template and cloned between the attL1 and attL2 sites in the Gateway entry vector pENTR/D-TOPO (Invitrogen, http://www.invitrogen.com/) resulting in ARL2pENTR. Subsequent LR recombination (Invitrogen) into the binary vector pK7FWG2 (Karimi et al., 2002) resulted in 35S::ARL2-GFP, which fuses GFP to the C-terminus of ARL2 under the control of the 35S promoter. For the proARL2::ARL2-GFP construct, the complete genomic sequence of ARL2 without the stop codon, as well as 2354 kb of upstream sequence, was PCR-amplified from Col-0 genomic DNA using the primers 5′-CACCTAATAGTATAAATGTTCAGTCAGAGTT-3′ and 5′-GTTTTTTTTCTTGTCTTGCCTCAGC-3′, and cloned into pENTR/D-TOPO, resulting in proARL2::ARL2pENTR. The insert was then recombined into pK7FWG2w/o35S (a pK7FWG2 plasmid that had been modified by removal of the 35S promoter as an SpeI–SacI fragment, followed by blunt-ending the remaining vector using Klenow fragment, and ligation). The DNA sequence of each construct was confirmed by sequencing. Transformation of Arabidopsis using Agrobacterium tumefaciens strain GV3101 was performed by the floral dip method (Clough and Bent, 1998).

Histochemical staining for starch, cell walls, membranes and GUS activity

For starch staining, 4- or 5-day-old seedlings were immersed in 1% lugol solution (0.1% potassium iodine, 0.05% iodine, in distilled water) for 1–2 min, and mounted on slides in water saturated with chloral hydrate. For GUS activity, vertically grown or gravi-stimulated seedlings were immersed in 100 mm sodium phosphate, pH 7.2, containing 1.9 mm 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (X-Gluc, Research Organics; http://www.resorg.com), 2 mm potassium ferricyanide and 0.5% Triton X-100. Seedlings in solution were subjected to 3.3 kPa vacuum for 30 min, followed by incubation for up to 16 h at 37°C. Samples were examined with a Leica DM LB2 microscope (Leica; http://www/leica-microsystems.com) using DIC/Nomarski optics, and images were recorded with a CCD camera. For cell-wall and membrane staining, 3–4-day-old seedlings were immersed in 10 μg ml−1 propidium iodide (Sigma, http://www.sigmaaldrich.com/) for 5–10 min, or 10 nM FM4-64 (Molecular Probes; http://www.probes.invitrogen.com) for 2 min, respectively, mounted in water and examined by laser scanning confocal microscopy using 488 nm excitation light from an argon laser and a 560 nm long-pass (LP) emission filter.

Antibodies and in situ immunolocalization

In situ immunolocalization of PIN3 was performed as described previously (Young et al., 2006). Determination of bias in lateral localization (Table 2) was performed by analysis of the entire z-series for each root. The sample genotype and direction of stimulation were not known at the time of evaluation in order to eliminate subjective interpretation. Individual roots were recorded as presenting a bias in PIN3 protein localization if several of their columella cells displayed preferential PIN3 localization on the same side (upper or lower) relative to the general axis of the root. In situ immunolocalization of PIN2 and α-tubulin was performed as described previously (Boonsirichai et al., 2003) using 1:200 anti-AGR1/PIN2 (Boonsirichai et al., 2003) and 1:100 anti-α-tubulin (clone B-5-1-2, Sigma). Rhodamine-conjugated anti-rabbit (Sigma; http://www.sigmaaldrich.com) and Cy3TM-conjugated anti-mouse (Jackson Labs) were used as secondary antibodies at 1:300. For Western blots, anti-GFP (Davis and Vierstra, 1998) was used at 1:8000 and anti-PUX1 (Rancour et al., 2004) and anti-PEP12 (da Silva Conceição et al., 1997) were used at 1:5000. Detection of positive signals was performed using HP-conjugated anti-rabbit at 1:20 000 (Sigma).

Fluorescence microscopy and plasmolysis

Laser scanning confocal micrographs were generated using either a Bio-Rad MRC-1024 (http://www.bio-rad.com/) or a Zeiss LSM510 microscope (http://www.zeiss.com/) at the Keck Biological Imaging Laboratory, University of Wisconsin-Madison. Multi-photon images were generated using a Bio-Rad Radiance 2100 MP Rainbow multi-photon system at Keck Biological Imaging Laboratory. Plasmolysis was performed by immersing 3-day-old seedlings in 0.8 m mannitol, 1 m potassium nitrate or water (control) containing counterstain (see Figure 5) for 6 min, mounting in solution without stain, and observing by confocal microscopy.

RT-PCR

cDNA was generated using SuperScript III reverse transcriptase (Invitrogen) from total RNA, isolated using an RNA-easy kit (Qiagen, http://www.qiagen.com/) from approximately 100 mg of 5-day-old whole seedlings grown on nutrient medium. Equal amounts of cDNA were then used to perform PCR using the following primer pairs: 5′-CGAGAAGATGAGCGCGAAAAAGCTTGAA-3′ and 5′-TATATCATCAATCCACCATCACAAT-3′ for ARG1, 5′-CACTCAAACTGGGAAACAAAAAC-3′ and 5′-TTCAACGCCTCCCCCAATAAATTT-3′ for ARL2, and 5′-CTCAGATCGAAATCGAGAAGAAG-3′ and 5′-GAACCGGAAGCAGACAGAAG -3′ for PAE2, for 30 cycles (ARG1), 40 cycles (ARL2) or 25 cycles (PAE2). The mutant and wild-type cDNAs from ARL2 and arl2-4 were cloned into pGEM-T and sequenced. Basic molecular protocols were as described by Sambrook et al. (1989).

Membrane preparation

Membrane fractions were prepared as described previously (Boonsirichai et al., 2003), except that plants were 1-week-old, the buffer lacked DTT, and membranes were pelleted for 30 min.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank David Rancour (Department of Biochemistry, UW-Madison, USA) and Scott Saracco for assistance with biochemical procedures, Sebastian Bednarek (Department of Biochemistry, UW-Madison, USA) for PUX1 and PEP12 antibodies, Richard Vierstra for GFP antibodies, Lance Rodenkirch (W.M. Keck Laboratory for Biological Imaging, UW-Madison, USA) for assistance with microscopy, Jiri Friml (Center for Plant Molecular Biology, University of Tübingen, Germany) for pin3-3 and DR5rev::GFP seed, Jessica Will for her diligent technical assistance, and John Stanga for comments on the manuscript and help with two of the figures. This work was supported by the National Science Foundation (grants MCB-0240084 and IOS-0642865). This is manuscript number 3637 of the Laboratory of Genetics, University of Wisconsin-Madison.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1.arl2-4 and arg1-3 are null alleles

Figure S2. PIN2 localization is not affected in arg1-2 and arl2-3 mutant roots.

Figure S3. Correlation between auxin reporter expression and starch staining of cell files.

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