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First published online May 26, 2006; 10.1104/pp.106.077818 Plant Physiology 141:966-976 (2006) © 2006 American Society of Plant Biologists RPA, a Class II ARFGAP Protein, Activates ARF1 and U5 and Plays a Role in Root Hair Development in Arabidopsis1,[W]Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (X.-F.S., C.-Y.Y., J.L., W.-C.Y.); and Graduate School of the Chinese Academy of Sciences, Beijing 100039, China (X.-F.S., C.-Y.Y.)
The polar growth of plant cells depends on the secretion of a large amount of membrane and cell wall materials at the growing tip to sustain rapid growth. Small GTP-binding proteins, such as Rho-related GTPases from plants and ADP-ribosylation factors (ARFs), have been shown to play important roles in polar growth via regulating intracellular membrane trafficking. To investigate the role of membrane trafficking in plant development, a Dissociation insertion line that disrupted a putative ARF GTPase-activating protein (ARFGAP) gene, AT2G35210, was identified in Arabidopsis (Arabidopsis thaliana). Phenotypic analysis showed that the mutant seedlings developed isotropically expanded, short, and branched root hairs. Pollen germination in vitro indicated that the pollen tube growth rate was slightly affected in the mutant. AT2G35210 is specifically expressed in roots, pollen grains, and pollen tubes; therefore, it is designated as ROOT AND POLLEN ARFGAP (RPA). RPA encodes a protein with an N-terminal ARFGAP domain. Subcellular localization experiments showed that RPA is localized at the Golgi complexes via its 79 C-terminal amino acids. We further showed that RPA possesses ARF GTPase-activating activity and specifically activates Arabidopsis ARF1 and ARF1-like protein U5 in vitro. Furthermore, RPA complemented Saccharomyces cerevisiae glo3  gcs1 double mutant, which suggested that RPA functions as an ARFGAP during vesicle transport between the Golgi and the endoplasmic reticulum. Together, we demonstrated that RPA plays a role in root hair and pollen tube growth, most likely through the regulation of Arabidopsis ARF1 and ARF1-like protein U5 activity.
The polar growth of cells as exemplified in root hairs and pollen tubes is a common phenomenon in plants. The tip growth of root hairs and pollen tubes is due to the deposition of cell membranes and wall materials at a restricted tip area of the plasma membrane (Schnepf, 1986  ). Recent genetic studies in Arabidopsis (Arabidopsis thaliana) have shown that small GTPases, such as ADP-ribosylation factors (ARFs), Rho-related GTPases from plants (ROPs), and Ras-related in brain, play important roles during the polar growth of root hairs and pollen tubes (Zheng and Yang, 2000; Cheung et al., 2002; Gu et al., 2004; Preuss et al., 2004). For example, ROP2 GTPase is a positive regulator of both root hair initiation and tip growth. Dominant-negative GDP-bound ROP2 reduces the number of hair-forming sites and leads to shorter, wavy hairs, whereas the expression of constitutively active GTP-bound ROP2 inhibits and depolarizes root hair tip growth. ROP2 overexpression results in longer root hair with multiple tips (Jones et al., 2002). Similarly, ARF1, a Golgi-localized protein, is required for root hair tip growth by modulating ROP2 function in Arabidopsis (Xu and Scheres, 2005). In addition, ARF1 also affects the localization of the auxin efflux carrier PINFORMED2 (PIN2) with slow kinetics, resulting in changes in auxin polar transport (Xu and Scheres, 2005).
Vesicular transport in mammalian cells and yeast (Saccharomyces cerevisiae) is regulated by a large family of RAS-related GTPases (Novick and Zerial, 1997
ARFGAPs, characterized by the presence of a zinc-finger motif and a conserved Arg residue within the ARFGAP domain, consist of a large gene family with 15 members in Arabidopsis (Vernoud et al., 2003
Identification of Mutants in Putative ARFGAP Genes
To study the role of ARFGAP in plant development, we identified a mutant sgt3118 in which the Dissociation (Ds) element was inserted into a putative ARFGAP gene, At2g35210 (also named AtAGD10; Vernoud et al., 2003
The full-length cDNA of At2G35210 is 1,475 bp in length with an open reading frame (ORF) of 1,188 bp (Fig. 1B); it encodes a peptide of 395 amino acids (Fig. 1B) with a molecular mass of 43.08 kD and a pI of 9.57. Sequence comparison of the cDNA and genomic fragment revealed that there are seven exons and six introns in the AT2G35210 gene. In AT2G35210, an N-terminal CXXCX16CXXC motif in the ARFGAP domain represents a typical zinc-finger structure (Fig. 1B), which is also present in the PF01412 family of plants (http://www.sanger.ac.uk/cgi-bin/Pfam/getacc?PF01412), ARFGAP1 in rats, and GROWTH COLD SENSITIVE1 (GCS1) and GCS1P-LIKE ORF3 (GLO3) in yeast (Cukierman et al., 1995 ). In the ARFGAP region, there are three REV interacting domains (residues 22–41, 41–58, and 62–83). In addition, there is one domain, GAKKTGKT, that matches the consensus [A/G]-X4-G-K-[S/T] of the potential ATP-/GTP-binding motif (the P-loop; PS00017; Fig. 1B).
Sequence analysis revealed that AT2G35210 belongs to the class II ARFGAP family with six members (AT5G54310, AT3G53710, AT2G37550, AT4G17890, AT5G46750, and AT2G35210; also named AtAGD5–10) in Arabidopsis (Vernoud et al., 2003
To determine the expression pattern of AT2G35210 in Arabidopsis, reverse transcription (RT)-PCR analysis was performed. AT2G35210 expression was detected strongly in inflorescences and at a lower level in roots (Fig. 2A ). This result indicated that AT2G35210 is specifically expressed in flowers and roots, which suggests that AT2G35210 might play a role in flower and root development.
To determine the cellular expression pattern of AT2G35210, Pro:AT2G35210-  -glucuronidase (GUS):Ter was constructed and transformed to Arabidopsis. Seedlings, roots, leaves, and inflorescences of independent transgenic lines were checked for GUS activity. As a result, GUS activity was observed specifically in mature pollen grains (Fig. 2, B and C) and pollen tubes within the style (Fig. 2D). In addition, GUS activity was also detected in roots, especially in root hairs (Fig. 2, E and F), lateral root primordia (Fig. 2F), and lateral roots (Fig. 2G). No GUS activity could be detected in other tissues (data not shown). The GUS pattern is consistent with RT-PCR analysis. These data indicated that AT2G35210 is expressed in pollen grains, pollen tubes, and roots, including root tips and root hairs. Therefore, we designated AT2G35210 as RPA.
Because ARFGAP is a key regulatory component in vesicle formation for membrane transport, we investigated whether RPA protein is involved in the secretory system. The P35S:RPA-enhanced green fluorescent protein (EGFP) construct and the P35S:EGFP control vector were introduced into Arabidopsis via Agrobacterium-mediated transformation. Confocal laser-scanning microscopy on transgenic root hairs showed that RPA-EGFP was detected as punctuate structures scattered within the cytoplasm (Fig. 3A
), whereas the EGFP control showed homogeneous localization. To determine the precise subcellular localization of RPA-EGFP, transgenic roots were stained with the Golgi marker BODIPY FL C5 ceramide (B–22650; Molecular Probes; Fig. 3B). Confocal laser-scanning microscopy showed that most of the RPA-EGFP fluorescence colocalized with the Golgi marker (Fig. 3C). These results suggested that RPA mainly resides in or associates with the Golgi apparatus. To further confirm its Golgi localization, the P35S:RPA-EGFP construct was cobombarded to onion (Allium cepa) epidermal cells together with the P35S:N-sialyltransferase (N-ST)-monomeric red fluorescent protein (mRFP) construct coding for the Golgi-localized N-ST fused to mRFP (Lee et al., 2002
To further study which part of the RPA protein determines its subcellular localization in the cell, we fused three truncated RPA peptides: RPA128 (residues 1–128), RPA246 (1–246), and RPA316 (1–316; Fig. 3G) to the N terminus of EGFP, respectively, and introduced them into Arabidopsis. The fluorescence of the three truncated RPA proteins was distributed throughout the whole cell, including the nucleus (Fig. 3, H–J). These results indicated that all three truncated RPA proteins lacking the 79 C-terminal amino acids could not be targeted to Golgi complexes correctly. This suggests that the 79 C-terminal residues of the RPA are responsible for its Golgi localization in the cell. To confirm this possibility, the 79 C-terminal amino acids of RPA were fused to EGFP to yield the P35S:RPAC79-EGFP construct, which was then cobombarded with the P35S:N-ST-mRFP construct to onion epidermal cells. As we expected, RPAC79-EGFP colocalized with N-ST-mRFP (Fig. 3, K–M), demonstrating that the Golgi localization of RPA protein was indeed determined by its 79 C-terminal amino acids.
To investigate whether RPA has ARF GTPase-activating activity as suggested by its homology to mammalian ARFGAP1, an in vitro ARF GTPase-activating activity assay was carried out with its potential substrates. The selection of putative ARF substrates was made based on their subcellular localization, expression pattern, and sequence homology. Among the ARF proteins, ARF1, ARF2, and ARF3 are associated with the Golgi complexes. ARF1 and ARF3 represent different phylogenetic branches of the ARF family and have been used previously for ARFGAP assays. In addition, several ARF-like proteins have been predicted to play a role in vesicle trafficking. Therefore, we chose ARF1, ARF3, and ARF-Likes (ARLs) as potential substrates for RPA. We first analyzed their expression pattern with RT-PCR. All of them showed a broad expression pattern throughout the plant, with higher expression levels in roots and inflorescences, except U2 (Fig. 4 ). At least their expression pattern overlaps with RPA in inflorescences and roots.
To further narrow down the number of candidate substrates, we used the stable GTP-binding property of ARFs (Ding et al., 1996 ) to facilitate our substrate selection. We fused the N terminus of ARF1, ARF3, U2, and U5 proteins to a poly-His tag and produced in Escherichia coli because it was difficult to obtain soluble, full-length recombinant proteins. Such truncated forms of ARFs have previously been used successfully in GAP activity assays in humans (Paris et al., 1997) and Arabidopsis (Rikke et al., 2000). In addition, His-6-RPA was also produced in E. coli for the assay. The GTP-binding assay of the in vitro-expressed His-6-ARF117, His-6-ARF317, His-6-U517, and His-6-U216 fusion proteins was performed. The result showed that ARF1 and U5 have strong GTP-binding capability (Fig. 5A
), ARF3 has very weak GTP-binding activity (Fig. 5A), and U2 and RPA have no detectable GTP-binding capability (Fig. 5A). Finally, ARF1, ARF3, and U5 were used for the ARFGAP assay.
ARF GTPases do not exhibit intrinsic GTPase activity (Kahn and Gilman, 1986 ) and, therefore, rely on the GTPase-activating activity of ARFGAPs to switch from the active GTP-bound form to the inactive GDP-bound form. To investigate whether RPA possesses GTPase-activating activity that mediates effective ARF1-GTP, ARF3-GTP, and U5-GTP hydrolysis, His-6-ARF117, His-6-ARF317, and His-6-U517 were first incubated with [ -32P]GTP, respectively, for 30 min, then His-6-RPA protein was added and incubated for another 15 or 30 min. As the incubation time and amount of His-6-RPA protein increased, the total [-32P] bound to putative substrate His-6-ARF117 declined (Fig. 5B); this result suggested that the GTP bound to the substrate was hydrolyzed by His-6-RPA protein and the hydrolysis is dependent on the presence of the protein in a dosage- and time-dependent manner. Similarly, His-6-RPA protein could also activate the GTPase activity of the His-6-U517 (Fig. 5C) with similar efficiency as that to His-6-ARF117. However, His-6-RPA protein could not activate His-6-ARF317 (data not shown). In a control experiment with either RPA alone or GTP as a substrate, only a residual amount of radioactivity was retained on the membrane (Fig. 5, B and C). These data demonstrated that RPA is indeed able to activate ARF1 and the ARF1-like protein U5 GTPase activity in vitro.
Previous studies showed that GLO3 and GCS1 are two ARFGAPs of yeast. In vitro assay indicated that efficient retrieval transport from the Golgi to the ER requires these two proteins (Poon et al., 1996
Root Hair Development Was Altered in the rpa Mutant To investigate the biological function of the putative ARFGAP gene, a phenotypic analysis was carried out on F2 individuals homozygous for the Ds insertion. The sgt3118 mutant seedlings, named rpa-1, displayed several aberrant root hair phenotypes, including bulged (Fig. 7, A, C, D , and E), branched (Fig. 7, E, F, and H), and shorter root hair (Fig. 7A) compared with that of the Landsberg erecta (Ler) wild type (Fig. 7B). With PCR analysis, we identified the homozygous SALK_000767 line, named rpa-2, and the root hairs of rpa-2 also showed bulged (Fig. 7, I and J), branched (Fig. 7, K and L), and shorter root hair (Fig. 7I). Furthermore, the root hair phenotype of rpa-1 could be rescued by the RPA gene (Fig. 7M). This evidence indicated that the root hair phenotype is indeed caused by the loss of function of the RPA gene.
To quantify these differences, we measured root hair length and width, root hair number per millimeter of root length, epidermal cell length, and branched root hair percent in rpa-1 and rpa-2 lines and their wild-type counterparts (Fig. 8 ). Microscopic observation showed that rpa-1 and rpa-2 have shorter root hairs than the wild type, with only one-seventh the length of wild-type root hairs (approximately 628 µm; Fig. 8A), whereas the width of both rpa-1 and rpa-2 are 2 times that of wild-type hairs (9.75 µm; Fig. 8B). When the number of root hairs per millimeter were measured, rpa-1 and rpa-2 have less hairs compared to that of the Ler wild type (approximately 31; Fig. 8C). The length of the mutant epidermal cells is about three-fourths of wild type for rpa-1 (Fig. 8D) and two-thirds for rpa-2 (approximately 186.7 µm; Fig. 8D); when the root hair density was corrected for epidermal cell length, rpa-1 still has about three-fourths of the root hairs of wild type (Fig. 8E), whereas rpa-2 has slightly more root hairs per epidermal cell length compared to that of Ler wild type. In addition, rpa-1 has about 23% branched hairs and rpa-2 has 6.47% branched hairs, whereas Ler wild type only has <1% branched hairs (Fig. 8F). These data indicate that rpa-1 has a slightly more severe phenotype than rpa-2 because rpa-2 is a weak allele where trace amounts of RPA transcripts were detectable, but not in rpa-1 (Supplemental Fig. 2). Together, the mutant phenotype of rpa-1 and rpa-2 suggests that RPA plays a role in root hair polar growth.
Pollen Tube Growth Is Slightly Affected in the rpa-1 Mutant Because the loss of RPA function affected the polar growth of root hairs, we investigated whether it also affected the polar growth of pollen tubes in rpa-1. After 8-h germination, about 25.0% for rpa-1 mutant pollen and 26.8% for Ler wild-type pollen had germinated. As the incubation time reached 16 h, the pollen germination rate reached 72.2% for rpa-1 pollen grains and 76.5% for Ler wild-type pollen grains. These results suggested that the loss of RPA function had no effect on pollen germination. To further investigate whether pollen tube polar growth was affected, the length of germinated pollen tubes was measured microscopically. There was little difference between Ler wild type and rpa-1 in pollen tube length at 8 h after germination and both were about 200 µm in length (Fig. 9 ). As the incubation time increased, the difference in pollen tube length between Ler wild type and rpa-1 became obvious. After 16-h incubation, the pollen tubes of Ler wild type were more than 700 µm in length and that of rpa-1 was only about 500 µm, 200 µm shorter than that of wild-type pollen tubes (Fig. 9). When the incubation time was extended to 24 h, the rpa-1 pollen tubes were 500 µm shorter than that of Ler wild type. This suggested that the loss of RPA function indeed had an effect on pollen tube elongation.
Taken together, we concluded that the root hair and pollen tube phenotypes were caused by loss of function of the RPA gene, suggesting that RPA plays a role in root hair development and pollen tube elongation.
RPA, a Functional ARFGAP of ARF1 and U5, Plays a Role in Root Hair Development in Arabidopsis
In this article, we described the phenotypic and molecular characterization of a novel ARFGAP in Arabidopsis. RPA belongs to the class II ARFGAP family (Vernoud et al., 2003
In Arabidopsis, 21 putative ARF and ARL GTPases and 15 ARFGAPs have been identified based on sequence homology (Vernoud et al., 2003
Recently, it was shown that ARF1 plays a role in the subcellular localization of the auxin efflux carrier PIN2 in root cells (Xu and Scheres, 2005
Our data showed that the RPA gene is highly expressed in flowers. GUS reporter analysis indicated that RPA is expressed in pollen grains and pollen tubes. Nevertheless, the homozygous rpa mutant is completely fertile and its pollen grains have the same competitiveness as wild type. A simple explanation would be genetic redundancy due to the presence of a large family of functionally overlapping genes. In vitro pollen germination assays showed that the rpa pollen tubes grow slower than wild type. Because the difference in growth becomes significant only after 12 h of germination, it is longer than the time (about 5–7 h) required for pollen tubes to reach ovules in Arabidopsis pistils. It is therefore reasonable to explain the fertile phenotype. Such defects in pollen tube growth may not be detrimental to plants having short styles, such as Arabidopsis, but may be detrimental to plants having long styles or long distances between ovules and the site of pollen germination, such as maize (Zea mays). Indeed, mutation in ROP2 does affect male competitiveness in maize, where pollen tubes have to travel a long distance to reach the ovules (Arthur et al., 2003
Plant Materials and Growth Conditions
Arabidopsis (Arabidopsis thaliana) ecotypes Ler and Columbia (Col) were grown on soil at 22°C ± 2°C in a greenhouse with a 16-h light/8-h dark cycle. Seeds were surface sterilized with 20% commercial bleach for 5 min, 70% ethanol for another 5 min, then rinsed four times with sterile water and germinated onto a Murashige and Skoog plate (Murashige and Skoog, 1962
Pollen grains were collected from freshly dehiscent anthers of Ler wild type and sgt3118 at the end of stage 12 of flower development (Smyth et al., 1990
Total RNA was extracted from roots, stems, leaves, inflorescences, siliques, and 7-d-old seedlings using TRIzol reagent (Invitrogen). One microgram of total RNA was used as a template and reverse transcribed with alfalfa mosaic virus reverse transcriptase (TaKaRa) in the presence of oligo(dT) primers. One microliter of the RT product was then used as a template in subsequent PCR reactions with 95°C for 1 min, 30 cycles of 94°C for 30 s, annealing for 30 s according to specified temperature, 72°C for 90 s, and, finally, 72°C for 10 min. PCR products were analyzed with 1% agarose gel.
For the construction of the Pro:RPA-GUS:Ter reporter gene, the 4.8-kb genomic fragment of the RPA was separated into three parts, amplified separately with primer pairs 3118P-5'/3118P-3', 3118G-5'/3118G-3', and 3118T-5'/3118T-3' (Table I ), then cloned into the binary vector pBI101 (CLONTECH) to give rise to the Pro:RPA-GUS:Ter fusion gene.
For the complementation test of the rpa-1 mutant, the RPA ORF was amplified by PCR with gene-specific primers and cloned downstream of the 35S promoter in the binary vector pGD1301 to yield the pGD1301-RPACDS construct. At the same time, the 4.8-kb genomic sequence of the RPA gene was also cloned into pGD1301, giving rise to the pGD1301-RPA construct. To produce proteins for biochemical assay, the RPA ORF was amplified by PCR with gene-specific primers RPA-EcoRI-5'/RPA-XhoI-3' (Table I) and then subcloned into the pET28b (Novagen) vector between EcoRI and XhoI to generate His-tagged RPA. Similarly, several potential substrates of RPA, such as ARF1, ARF3, U5, and U2, were selected and His-tagged proteins were produced in Escherichia coli. Briefly, the truncated version of ARF1 (18–181 amino acids), ARF3 (18–182 amino acids), U5 (18–165 amino acids), and U2 (17–185 amino acids) were amplified by PCR with primer pairs: ARF1-BamHI-5'/ARF1-XhoI-3', U5-BamHI-5'/U5-XhoI-3', U2-BamHI-5'/U2-XhoI-3' (Table I), respectively. The PCR products were subcloned into the pET28a expression vector between BamHI and XhoI; ARF3 (18–182 amino acids) was amplified with primer pair ARF3-BamHI-5'/ARF3-NotI-3' (Table I) and cloned into the pET28a vector between BamHI and NotI. The His-RPA protein was purified by a nickel column under denaturing conditions and renatured by dialysis. The His-tagged ARF1, ARF3, U2, and U5 proteins were purified by nickel-nitrolotriacetic acid spin columns under native conditions according to manufacturer's recommendation (Qiagen).
The ORF of RPA was obtained through RT-PCR with the gene-specific primer pair RPA-BamHI-5'/RPA-KpnI-3' (Table I) and cloned into the pEGFP vector (CLONTECH) between the BamHI and KpnI sites, then the RPA-EGFP fragment was subcloned downstream of the 35S constitutive promoter into pCAMBIA1301 between BamHI and XbaI to give rise to P35S:RPA-EGFP. Similarly, RPA128-EGFP, RPA246-EGFP, RPA316-EGFP, and RPAC79-EGFP were constructed with gene-specific primers (Table I). The plasmids P35S:RPA-EGFP and P35S:N-ST:mRFP were cobombarded into onion (Allium cepa) cells using the PDS-100/He Biolistik particle delivery system (Bio-Rad). The bombarded samples were incubated at 22°C for 24 h on Murashige and Skoog plates. Fluorescent signals were observed using the Zeiss LSM510 META laser-scanning microscope at the Center for Developmental Biology.
One microgram of gel-purified ARFs were spotted onto nitrocellulose membrane (Pall Gelman Science) and treated with hybridization solution (50 mM Tris-HCl, 5 mM MgCl2, 0.3% Tween 20, 0.5 mM EDTA, 5 mM dithiothreitol [DTT], and 0.3% bovine serum albumin, pH 7.5) for 30 min at 30°C and then transferred to new hybridization solution containing [
ARF GTPase-activating activity was detected according to Goldberg (1999)
The temperature-sensitive glo3 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AT2G35210.
We thank Professor G.C. Johnston (Dalhousie University, Halifax, Nova Scotia, Canada) for kindly providing the glo3 gcs1 strain and the YEP352 vector, and Professor Kang Chong (Institute of Botany, Chinese Academy of Sciences, China) for support with experimental materials; Professor Inhwan Hwang (Pohang University of Science and Technology, Korea) for providing us with P35S:N-ST-mRFP; and Dr. Dongqiao Shi (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing) for technical assistance in confocal microscopy. We also thank the Arabidopsis Biological Resource Center, Ohio State University, for providing the T-DNA insertion lines. Received January 25, 2006; returned for revision May 12, 2006; accepted May 12, 2006.
1 This work was supported by the BAI REN JI HUA program, Chinese Academy of Sciences, and the National Science Foundation of China (grant no. 30425030). 
2 These authors contributed equally to the paper. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Wei-Cai Yang (wcyang{at}genetics.ac.cn).
[W] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.077818. * Corresponding author; e-mail wcyang{at}genetics.ac.cn; fax 86–10–62551272.
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ABSTRACT
RESULTS
-32P-GTP-ARF1. RPA, No ARF1 protein added; GTP, no protein added. C, GTPase-activating activity of RPA on U5 protein assessed by hydrolysis of GTP bound to U5. RPA, No U5 protein added; GTP, no protein added.
