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First published online January 19, 2005; 10.1105/tpc.104.028449 © 2005 American Society of Plant Biologists
Dissection of Arabidopsis ADP-RIBOSYLATION FACTOR 1 Function in Epidermal Cell Polarity
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INTRODUCTION |
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). Genetic screens have revealed specific regulators of vesicle trafficking in animal and plants (see below), but core factors, such as ADP-ribosylation factors (ARFs), have mainly been studied in cultured cells.
ARF family members cycle between an active GTP-bound and an inactive GDP-bound state. A subset of ARF proteins is referred to as class 1 based on their sequence conservation, and these proteins regulate intracellular vesicular trafficking at multiple stages of the secretory and lysosomal/vacuolar transport pathways in mammalian and plant cells (Balch et al., 1992; Lee et al., 2002; Presley et al., 2002; Takeuchi et al., 2002; Pimpl et al., 2003). In particular, class 1 ARF proteins participate in the formation of transport vesicles and the selection of transmembrane protein cargo from donor compartments in mammalian cells (Goldberg, 2000; Spang, 2002).
The vesicle transport inhibitor brefeldin A (BFA) has been shown to interfere with ARF action in mammalian, fungal, and plant cells (Chardin and McCormick, 1999; Nebenfuhr et al., 2002). BFA inhibits the rate of guanine nucleotide exchange exhibited by ARF1 and interferes with ARF1 function in COPI coat recruitment. Crystal structure of the ARF1-GDP/Sec7/BFA complex shows that BFA binds at the protein–protein interface to inhibit conformational changes in ARF1 required for Sec7 to dislodge the GDP molecule (Mossessova et al., 2003). BFA induces redistribution of Golgi proteins into the endoplasmic reticulum (ER) in mammalian and plant cells (Lippincott-Schwartz et al., 1989; Saint-Jore et al., 2002). Moreover, interference of BFA with the endocytic pathways in animals and plants has been reported (Lippincott-Schwartz et al., 1991; Geldner et al., 2003; Grebe et al., 2003). BFA also interferes with cell polarity in plants, in which it disturbs zygote polarity in Fucus (Shaw and Quatrano, 1996), randomizes the root hair initiation site of Arabidopsis thaliana trichoblast cells, and interferes with the localization of putative auxin efflux carrier components of the PIN family and auxin influx carrier AUX1 (Geldner et al., 2001; Grebe et al., 2002).
Consistent with a potential role for the ARF proteins in the establishment of plant cell polarity, mutations in BFA-sensitive Arabidopsis GNOM/EMB30 ARF-GEF (guanine nucleotide exchange factor) lead to aberrant cell shape and orientation of cell division (Mayer et al., 1993; Shevell et al., 1994). GNOM/EMB30 protein localizes to endocytic organelles, and one of its functions is to control the polarized trafficking of the BFA-sensitive auxin efflux component PIN1 to the basal plasma membrane (Steinmann et al., 1999; Geldner et al., 2003).
Additional targets for ARF-dependent vesicle trafficking are suggested by the observation that BFA inhibits early polar localization of ROP (RHO of plants) proteins at the root hair initiation site (Molendijk et al., 2001). ROP proteins are homologs of the RHO/RAC/CDC42, members of the RHO family GTPases, which include key regulators of cell polarity in yeast and animals (Etienne-Manneville and Hall, 2002). ARF family GTPases are required for several CDC42-mediated functions because human CDC42 is BFA sensitive and expression of a dominant-negative form of human ARF1 results in the redistribution of Golgi-localized human CDC42 to the cytosol (Erickson et al., 1996).
In comparison with yeasts and animals, Arabidopsis has many class 1 ARF proteins (Jürgens and Geldner, 2002; Vernoud et al., 2003). Transient expression of constitutively active (GTP-locked form) or constitutively inactive (GDP-locked form) mutants of ARF1 in tobacco (Nicotiana tabacum) and Arabidopsis cultured cells shows that this protein is required for the sequence-specific vacuolar sorting route to the lytic vacuole (Pimpl et al., 2003) and influences ER-to-Golgi transport as well as Golgi-derived transport to the plasma membrane, dependent on the cargo molecule (Lee et al., 2002; Takeuchi et al., 2002). Moreover, Arabidopsis ARF1 is able to complement and rescue the ARF1-ARF2 lethal yeast double mutant (Takeuchi et al., 2002). These results suggest that class 1 ARF proteins in Arabidopsis have similar cellular functions to their yeast and animal counterparts.
Here, we investigate developmental functions of Arabidopsis ARF1. As our expression and loss-of-function analysis suggests extensive redundancy, we expressed GTP- and GDP-locked mutants of ARF1 using region-specific and heat shock–inducible promoters and analyzed the consequences in the context of the multicellular organism. We demonstrate that class 1 ARF function is essential in plant development and that regulated expression of GTP- and GDP-locked mutants of ARF1 can be used to demonstrate roles of ARF1 in apical-basal polarity of epidermal cells and for ROP and PIN protein localization.
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T-DNA insertion lines from the SIGNAL and the Syngenta GARLIC collections (McElver et al., 2001; Alonso et al., 2003; data not shown) revealed no obvious phenotypes, indicating extensive functional redundancy. Multiple RNA interference lines targeted to a common class 1 ARF sequence also revealed no specific phenotypes (data not shown).
If class 1 ARF proteins perform overlapping functions in Arabidopsis development, they should also share expression domains. We therefore created promoter fusions for all six class 1 ARF proteins using the ER-localized yellow fluorescent protein (ERYFP) as a reporter. For each promoter fusion, 20 independent transgenic lines were analyzed by confocal laser scanning microscopy (CLSM). ERYFP expression was observed in all the promoter fusion lines except for ARF6 (At5g14670), which is preferentially expressed in anthers and pollen (see Supplemental Figure 1A online). The promoter fusion lines for the other five class 1 ARF proteins showed essentially the same expression pattern with constitutive ERYFP expression in young seedlings (see Supplemental Figures 1B to 1F online), indicating extensive overlap of class 1 ARF gene expression.
We focused our studies on the Arabidopsis ARF1 (At2g47170) because this member has been studied at the cellular level (Lee et al., 2002; Takeuchi et al., 2002; Pimpl et al., 2003). GenBank EST expression data (http://www.ncbi.nih.gov/Genbank/) confirm that ARF1 is expressed in all organs examined. To investigate tissue specificity of ARF1 mRNA expression in different organs and at different developmental stages, we created ARF1 promoter fusions using ß-glucuronidase (GUS) as a reporter. GUS activity was detected throughout the roots of all the transgenic lines examined (see Supplemental Figure 1G online). In addition, we also observed strong GUS activity in vascular bundles, leaf primordia, stomata, trichomes, style, stigmatic tissue, and young pollen (see Supplemental Figures 1H to 1J online). Intense GUS activity was detected in embryo sacs and dissected embryos of all stages (see Supplemental Figure 1K online).
We investigated whether posttranscriptional regulation affected ARF1 expression by making stable transgenic Arabidopsis plants carrying the fusion construct of wild-type ARF1 (ARF1-WT) and enhanced green fluorescent protein (EGFP) under control of the Arabidopsis ARF1 promoter (arf1-ARF1-WT-EGFP). Constitutive GFP expression was found in all the ARF1-WT-EGFP translational fusion lines. For example, GFP fluorescence was observed in all cells within the root meristem, and GFP fluorescence localized to the region of forming cell plate, to the growing root hairs, and also was detected in stomata and trichome cells (see Supplemental Figures 1L to 1P online). Collectively, our data suggest that the ARF1 protein is ubiquitously expressed and overlaps with the expression domain of the class 1 ARF members.
Arabidopsis ARF1 Localizes to Golgi Apparatus and Endocytic Organelles
In both yeast and mammalian cells, ARF1-Q71L (GTP-locked form) has significantly reduced GTPase activity and therefore behaves as a constitutively activated mutant (Pepperkok et al., 2000) that cannot hydrolyze its GTP and interferes with the sorting of membrane proteins into Golgi-derived COPI vesicles. ARF-T31N (GDP-locked form) has low affinity for GTP and has been shown to behave as a dominant-negative mutant (Dascher and Balch, 1994) that blocks the formation of COPI-coated vesicles. As the critical amino acids are conserved among class 1 ARF proteins (data not shown), both mutants are expected to also impair ARF1-mediated vesicle transport in plants. We made protein fusion constructs of ARF1-WT and the GTP- and GDP-locked dominant mutants of ARF1, namely ARF1-Q71L and ARF-T31N with EGFP, enhanced cyan fluorescent protein (ECFP), or enhanced yellow fluorescent protein (EYFP), under control of a 35S promoter of Cauliflower mosaic virus (CaMV). We studied the intracellular localization and dynamics of the translocation of ARF1 protein during transient expression in onion (Allium cepa) epidermal cells. GTP-locked ARF1 colocalized with wild-type ARF1 in multiple punctuate structures, whereas no fluorescent motile organelles were observed in cells with GDP-locked ARF1 construct (Figures 1A to 1D; see Supplemental Movie 1 online).
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-2,6-sialyltransferase (N-ST) fused to YFP (N-ST-YFP; Grebe et al., 2003) or a Golgi-targeted N-acetylglucosaminyl (NAG) transferase fused to ECFP (NAG-ECFP) suggest that ARF1 largely resides in or associates with the Golgi apparatus but also localizes to organelles negative for the Golgi markers (Figure 1E; see Supplemental Figure 2A and Movies 2 to 4 online). The Golgi localization of ARF1 is in agreement with observations on maize (Zea mays) and tobacco cells using ARF1 antisera (Pimpl et al., 2000; Ritzenthaler et al., 2002). However, our data indicate that ARF1 localizes to additional sites. Indeed, we found that ARF1-WT-EGFP fluorescence partially overlaps with the endocytic marker FM4-64 (Figure 1F; see Supplemental Movie 5 online), which does not label Golgi apparatus in both onion and Arabidopsis cells (see Supplemental Figures 2B, 2D, and Movie 6 online), suggesting that Arabidopsis ARF1 is also localized to endocytic organelles. Furthermore, a subpopulation of ARF1 fluorescence coincides with a red fluorescent protein (RFP) fusion to the conventional type RAB5 homolog ARA7 from Arabidopsis (RFP-ARA7; Figure 1G; see Supplemental Movie 7 online) but not with a plant unique RAB GTPase ARA6 fused to EYFP (ARA6-EYFP; Ueda et al., 2001; see Supplemental Figure 2C and Movie 8 online), indicating that ARF1 and ARA7 reside in different endocytic organelles from ARA6. In addition, we found that ARF1-EYFP fluorescence moves along actin filaments, illustrated by an EGFP fusion to the mouse F-actin–binding protein TALIN (EGFP-TALIN; Kost et al., 1998; Figure 1H; see Supplemental Movie 9 online).
We next studied the intracellular localization of wild-type ARF1 and its mutated forms in transgenic Arabidopsis plants. A heat shock–inducible promoter from the Arabidopsis HSP18.2 gene (Takahashi et al., 1993) was used to drive expression of the fusion proteins. Intense GFP expression was induced in Arabidopsis root cells within 2 h after heat shock induction (37°C, 2 h). Both heat shock–induced ARF1-WT-YFP and ARF1-Q71L-YFP distribute similar to arf1-ARF1-WT-EGFP (Figures 2A, 2B, and 2D1), suggesting that the heat shock promoter activity levels used did not alter protein localization. In hsp18.2-ARF1-T31N-ECFP lines, CFP fluorescence is largely cytosolic and localizes to the nucleus, whereas no moving organelles were labeled (Figure 2C). Colocalization analysis of ARF1-WT-GFP with Golgi and endocytic markers in Arabidopsis confirms our observations in onion epidermal cells (Figures 2D and 2E; see Supplemental Figures 2D and 2E online), and in addition ARF1-WT-GFP coaggregates with FM4-64 in BFA-induced compartments (Figure 2F). Taken together, our data suggest that ARF1 resides both in Golgi stacks and in a subpopulation of vesicles that move along actin filaments.
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; Figures 4F and 4I), indicating that ARF-dependent processes are involved in apical-basal polarity and polar tip growth. In a search for specific ARF1 effects, we focused on polarity aspects of a single cell type: the trichoblast.
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) was used for region-specific expression of wild-type ARF1 and its mutated forms. Manipulation of ARF1 function in the root meristem using the root meristem–specific rch1GV driver affected root development, directional cell expansion, and cytokinesis (see Supplemental Figure 3 online), consistent with the expected household role of ARF1 in vesicle trafficking. However, it did not cause any obvious defects in root hair initiation and root hair tip growth (see Supplemental Figures 3C, 3F, 3H, and 3J online). We therefore specifically interfered with ARF1 function in trichoblasts. ARF1-WT and ARF1 mutants were expressed using a trichoblast-specific driver prp3GV whose expression is initiated at the onset of root hair outgrowth (Bernhardt and Tierney, 2000; Figure 3A). No alteration in apical-basal epidermal polarity or tip growth occurred in ARF1-WT–overexpressing lines (Figures 3B, 3G, 3L, and 3M), but trichoblast-specific expression of ARF1-Q71L or ARF1-T31N significantly inhibited tip growth (Figures 3C to 3F, 3H to 3K, 3N, and 3O).
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Consistent with the effect of BFA treatment (Grebe et al., 2002), apical-basal polarity in root hair initiation is dramatically affected by heat shock–induced expression of ARF1-T31N but not ARF1-Q71L (Figures 4G, 4H, and 4J). The difference in effect of the GTP- and GDP-locked mutants of ARF1 on epidermal polarity might indicate that they act on different targets, but can also reflect differences in protein level or stability. Apical-basal trichoblast polarity was not affected by the ARF1 mutants expressed in PRP3 or RCH1 expression domain as well as in the root cap region (data not shown). We concluded that root hair positioning in response to epidermal polarity and the decision to grow out a hair per se occur in the elongation zone.
ARF1 Function Is Required for ROP2 Localization and Action
The Arabidopsis ROP2/ARAC4 gene has been implicated as a regulator of both apical-basal polarity in root hair initiation and polarized root hair tip growth. The ROP2 gene is expressed throughout root hair development, and ROP2 overexpression results in longer root hairs with multiple tips, whereas constitutively active GTP-bound ROP2 (ROP2-G14V) expression inhibits and depolarizes root hair tip growth (Jones et al., 2002). Transgenic lines carrying prp3GV and UAS-ROP2-G14V display the bulged root hair phenotype reported for 35S-ROP2 lines (Figures 5A to 5C), whereas no root hair phenotype was observed in transgenic lines carrying rch1GV and UAS-ROP2-G14V (data not shown).
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). To determine whether ARF1 function is involved in ROP2 action, we studied genetic interactions between ARF1 and ROP2 in trichoblast cells. We crossed UAS-ARF1-Q71L or UAS-ARF1-T31N into the transgenic lines carrying prp3GV and UAS-ROP2-G14V, and performed epistasis analysis in the F1 progeny. In all the F1 lines examined, root hairs showed tip growth defects but displayed no bulging (Figures 5D to 5I), indicating that ROP2 function in controlling polarized tip growth is ARF1 dependent. We next investigated the localization of the ROP2 protein in Arabidopsis plants carrying the fusion construct of EYFP and ROP2 (EYFP-ROP2) under the control of the ROP2 promoter. YFP fluorescence is highly enriched in the apical and basal plasma membranes of the root meristematic cells (Figure 5J), and consistent with its role in cell polarity, EYFP-ROP2 localized to the future site of root hair formation as well as to the tips of growing root hairs (Figure 5K). Moreover, EYFP-ROP2 partially colocalized with endocytic marker FM4-64 upon BFA treatment (Figures 5L to 5N), implying ARF-dependent ROP2 trafficking. Heat shock (37°C, 2 h)–induced overexpression of wild-type ARF1 had no obvious effects on the localization of the EYFP-ROP2 fusion protein (Figure 5O). However, in ARF1-Q71L– or ARF1-T31N–expressing lines, localization of the EYFP-ROP2 fusion protein was significantly altered within 20 h after heat shock induction, illustrated by strong internalization and reduced YFP fluorescence on the plasma membrane (Figures 5P and 5Q). These results suggest that ARF1 function is required for ROP2 localization.
ARF1-Q71L and ARF1-T31N Act Rapidly on Golgi Trafficking and FM4-64 Uptake but Slowly on PIN2 Localization
Delocalization of ROP2 after ARF1 manipulation was not clearly observed until 20 h after heat shock, which posed the question whether dominant ARF1 mutants act slowly on all ARF1-dependent processes or whether ROP2 localization defects might require intermediate steps. To assess whether heat shock–induced ARF1 mutants can rapidly affect known vesicular trafficking pathways in the intact plant, we analyzed changes in the distribution of Golgi markers, a functional auxin efflux component PIN2-EGFP fusion, and the endocytic marker FM4-64. As a control, we timed the delocalization response of these markers to ARF-GEF inhibition by BFA treatment.
No changes were observed in the distribution of any marker after the overexpression of ARF-WT at all heat shock periods used (Figures 6A, 6E, 6I, 6M, 6Q, and 6U). However, within 3 h of heat shock at 37°C, fluorescence of the two Golgi markers N-ST-YFP and NAG-EGFP was significantly altered in both ARF1-Q71L– and ARF1-T31N–expressing lines (Figures 6B, 6C, 6F, and 6G). Surprisingly, PIN2-EGFP was visibly internalized only after prolonged heat shock (Figures 6J, 6K, 6N, and 6O), suggesting that PIN2 exocytosis responds slowly to ARF1 manipulation.
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). BFA treatment affects the localization of Golgi markers within 15 min to 1 h (Figures 6D and 6H), consistent with the rapid changes observed upon ARF1 modulation. However, PIN2-EGFP, a slow responder to ARF1 modulation, responds rapidly to BFA (Figures 6L and 6P). More strikingly, the response to BFA of the endocytic marker FM4-64 differs from its response to ARF1 manipulation because BFA strongly interferes with endocytic trafficking of FM4-64 from endocytic organelles to the vacuole but not with FM4-64 uptake (Figure 6T and 6X). This might suggest that ARF1 modulates uptake through interaction with a BFA-insensitive ARF-GEF.
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DISCUSSION |
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; Sanderfoot et al., 2001). Yet, studies on ARF interactors and their inhibitors suggest that the ARF system serves such context-specific roles, as illustrated by recent investigations on the GNOM/EMB30 ARF-GEF and its link to polar auxin transport in planta (Geldner et al., 2003). Here, we use ARF1-WT and GTP- and GDP-locked dominant mutants of ARF1 (ARF1-Q71L and ARF1-T31N) to dissect roles of ARF1 in Arabidopsis. We show that manipulation of Arabidopsis ARF1 function in separate domains influences expected processes such as cytokinesis and tip growth. However, we also reveal specific modulation of apical-basal epidermal polarity in a distinct phase of development. At the molecular level, we show that the kinetics of inducible ARF responses can be used to separate direct and indirect effects on protein localization.
ARF1 has been shown to localize to Golgi apparatus in both mammalian and plant cells (Vasudevan et al., 1998; Pimpl et al., 2000; Ritzenthaler et al., 2002). Unexpectedly, we detected Arabidopsis ARF1 in endocytic organelles, suggesting that it plays a role in the trafficking of endocytic vesicles like non-plant ARF1 (Gaynor et al., 1998; Gu and Gruenberg, 2000). Consistent with this notion, uptake and trafficking of the endocytic marker FM4-64 is inhibited by both GTP- and GDP-locked forms of ARF1. Moreover, a subpopulation of Arabidopsis ARF1 coincides with the RAB5 homolog ARA7 but not with ARA6 (Ueda et al., 2001).
The effects of the ARF-GEF inhibitor BFA suggest that ARF-dependent vesicle trafficking plays an important role in the establishment of apical-basal epidermal polarity and in root hair tip growth (Grebe et al., 2002). Here, we show that dominant mutants of ARF1 interfere with both aspects of cell polarity, raising the question whether ARF1 is a target for both BFA-sensitive ARF-GEF–dependent polarity changes.
Which effectors are important for these polarization events? We show that polar ROP2 localization to the hair tip and phenotypic activity of ROP2 in this region require ARF1 function. These data functionally support the idea that root hair initiation involves ARF1-dependent polar localization of the ROP proteins (Molendijk et al., 2001). The slow relocalization of ROP2 after ARF1 manipulation suggests that intermediate steps are involved and that ARF1 may not be the principal ARF regulated by BFA-sensitive ARF-GEFs in ROP2 trafficking. However, we cannot exclude the formal possibility that these slow kinetics reflect interference of ARF1 manipulation with unrelated pathways.
The auxin transport machinery and actin cytoskeleton have been implicated in the establishment of apical-basal trichoblast polarity (Masucci and Schiefelbein, 1994; Sabatini et al., 1999; Grebe et al., 2002; Ringli et al., 2002). Furthermore, auxin influx and efflux carriers are BFA sensitive, and actin-mediated vesicle trafficking is required for the recycling of auxin efflux carriers (Morris and Robinson, 1998; Geldner et al., 2001, 2003; Grebe et al., 2002, 2003). We show that ARF1 manipulation can affect apical-basal polarity and interferes with PIN2-EGFP recycling, indicating that auxin transport carriers require proper ARF function. However, dominant ARF1 mutant–mediated PIN2-EGFP internalization is slow compared with changes in Golgi and endocytic markers, whereas PIN2-EGFP is among the fastest responders to BFA. These observations raise the possibility that ARF1 also influences vesicular cycling of auxin efflux carriers in an indirect way, whereas GNOM and/or other BFA-sensitive ARF-GEFs interact with another ARF member that is more directly involved in PIN recycling. Our findings suggest that ARF1 may not be the most BFA-sensitive target in polarization events involving ROP and PIN proteins, and they illustrate that caution needs to be exerted in inferring direct interactions from developmental phenotypes upon ARF manipulation.
Further dissection of specific ARF1-mediated processes will be required to reach a better understanding of its role in multiple distinct polarization events. A spectrum of interacting proteins, such as ARF-GEFs and ARF-GAPs, can provide specificity to ARF1 action. Furthermore, connected trafficking pathways may be secondarily affected, and these will have to be identified in each case. Our tissue-specific approach for ARF1 misregulation defines a ready-to-go system to search for specific vesicle transport components impinging on cell and tissue polarity by straightforward suppressor and enhancer screens.
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afb/vapster.html and imbibed for 4 d at 4°C in the dark, then germinated on vertically oriented plates containing half-strength MS agar (Duchefa, Haarlem, The Netherlands) and 1% sucrose, pH 5.8, and grown in a plant room at 22°C with a 16-h-light/8-h-dark cycle.
Plasmid Construction and Plant Transformation
ARF1 and ROP2 cDNA were amplified from an Arabidopsis cDNA library using PCR, and cDNA sequences were verified. Q71L and T31N mutations in ARF1 and the G14V mutation in ROP2 were introduced using a PCR-based site-mutagenesis strategy (Stratagene, La Jolla, CA).
For the arf1-ERYFP and arf1-GUS promoter fusion constructs, 2.64 kb of upstream sequence from the Arabidopsis ARF1 gene was fused to an ERYFP or GUS reporter gene and nopaline synthase terminator engineered in pGREENII-0229.
To make the arf1-ARF1-WT-EGFP, 35S-ARF1-WT-EGFP (or ECFP or EYFP), 35S-ARF1-Q71L-EGFP, 35S-ARF1-T31N-EGFP, hsp18.2-ARF1-WT-EGFP, hsp18.2-ARF1-Q71L-EYFP, and hsp18.2-ARF1-T31N-ECFP constructs, EGFP (or ECFP or EYFP) was fused in frame to the C terminus of ARF1 wild-type or mutant proteins. The resulting fusions were cloned into the pGREENII-0229 vector (http://www.pgreen.ac.uk) under the control of the Arabidopsis ARF1 promoter, CaMV 35S promoter, or Arabidopsis HSP18.2 promoter (847 bp), respectively.
NAG-EYFP and NAG-ECFP were generated from NAG-EGFP (Grebe et al., 2003) by exchanging of EGFP to EYFP or ECFP. For ARA6-EYFP, NAG cDNA was exchanged for ARA6 cDNA. To create EGFP-TALIN, the mouse TALIN F-actin–binding sequence from pSYC14 (a generous gift from N.H. Chua, Rockefeller University, New York, NY) was fused in frame to the C terminus of EGFP engineered in pGREENII-0229 between the CaMV 35S promoter and nopaline synthase terminator.
To create the rch1GV construct, 2.7 kb of upstream sequence from the Arabidopsis RCH1 gene (Casamitjana-Martinez et al., 2003) was fused to the GV coding sequence; the resulting construct also contained a UAS-ERGFP or UAS-GUS reporter engineered in the pGREENII-0029 vector. For prp3GV and hsp18.2GV constructs, 2 kb of Arabidopsis the PRP3 gene promoter region or the Arabidopsis HSP18.2 promoter sequence was subcloned before the GV coding sequence in the pGREENII-0029 vector with a UAS-GUS reporter. To generate UAS-ARF1-WT, UAS-ARF1-Q71L, UAS-ARF1-T31N, and UAS-ROP2-G14V plant transformation constructs, ARF1 cDNA, ARF1-Q71L, ARF1-T31N, and ROP2-G14V were subcloned into a UAS cassette engineered in pGREENII-0229, and these constructs were transformed directly into the GV lines.
For the EYFP-ROP2 protein fusion, the EYFP coding sequence was fused in frame to N terminus of the Arabidopsis ROP2 gene in pGREENII-0229 vector between 1.8 kb of the ROP2 promoter sequence and 400 bp of the ROP2 3'-untranslated region. PIN2-EGFP was generated by insertion of EGFP into PIN2 (Luschnig et al., 1998; Muller et al., 1998) genomic fragment (nucleotides –2157 to 4380 from ATG) at position 2312. Its functionality was verified by the complementation of the eir1-1 (pin2) agravitropic phenotype (Luschnig et al., 1998).
All the constructs were introduced into Agrobacterium tumefaciens strain C58C1 (GV3101) with the pSoup plasmid (http://www.pgreen.ac.uk) by electroporation. Plant transformation was done in Arabidopsis Columbia ecotype using the floral-dip method (Clough and Bent, 1998).
Histochemical Analysis of GUS Activity
Embryos and small seedlings were immersed in the staining solution (50 mM sodium phosphate buffer, pH 7.2, 0.1% Triton X-100, 1.5 mM potassium ferricyanide, 1.5 mM potassium ferrocyanide, and 1.5 mg/mL X-glucoronide) and incubated in the dark at 37°C. After staining, all the samples were cleared in 8:3:1 chloral hydrate:distilled water:glycerol and visualized using Nomarski optics on a Zeiss (Jena, Germany) Axioskop 2 microscope with a Nikon (Tokyo, Japan) DXM1200 digital camera.
Particle Bombardment–Mediated Transient Expression in Onion Epidermal Cells
For particle bombardment, all plasmids were amplified in Escherichia coli strain and purified using plasmid midi or mini kits according to the manufacturer's instructions (Qiagen, Valencia, CA). Onion (Allium cepa) bulb scale epidermis was bombarded with gold particles coated with plasmids using a Bio-Rad (Hercules, CA) PDS-1000/He particle delivery system. Bombarded specimens were incubated in water before CLSM analysis.
Inhibitor Treatments, FM4-64 Staining, and Analyses in Live Roots
BFA treatments were performed as described previously (Grebe et al., 2003). For FM4-64 stainings, onion epidermis or Arabidopsis seedlings were incubated in 2 mL of distilled water containing 5 µM FM4-64 for indicated periods at room temperature. Stained samples were washed twice with water and underwent heat shock treatment, or were directly observed by CLSM and/or two-photon LSM.
Microscopy
GFP, YFP, and CFP seedlings were visualized using a Leica (Wetzlar, Germany) MZ FLIII fluorescence stereomicroscope equipped with GFP and YFP filters. Propidium iodide (10 µg/mL in distilled water) was used to stain the cell walls of living root cells (red signal). Two-photon LSM and CLSM were used as described (Grebe et al., 2003).
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Acknowledgments |
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Footnotes |
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Online version contains Web-only data. 
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.104.028449.
Received October 9, 2004; accepted December 2, 2004.
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REFERENCES |
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