Skip to main content
NCBI home page
Journal of Experimental Botany logoLink to Journal of Experimental Botany
. 2013 Jul 11;64(11):3327–3338. doi: 10.1093/jxb/ert171

CPK3-phosphorylated RhoGDI1 is essential in the development of Arabidopsis seedlings and leaf epidermal cells

Yuxuan Wu 1,2, Shujuan Zhao 1,2, Han Tian 3, Yuqing He 1,2, Wei Xiong 1,2, Lin Guo 3, Yan Wu 1,2,*
PMCID: PMC3733153  PMID: 23846874

Abstract

The regulation of Rho of plants (ROP) in morphogenesis of leaf epidermal cells has been well studied, but the roles concerning regulators of ROPs such as RhoGDIs are poorly understood. This study reports that AtRhoGDI1 (GDI1) acts as a versatile regulator to modulate development of seedlings and leaf pavement cells. In mutant gdi1, leaf pavement cells showed shorter lobes in comparison with those in wild type. In GDI1-14 seedlings (GDI1-overexpression line) the growth of lobes in pavement cells was severely suppressed and the development of seedlings was altered. These results indicate that GDI1 plays an essential role in morphogenesis of epidermal pavement cells through modulating the ROP signalling pathways. The interaction between GDI1 and ROP2 or ROP6 was detected in the leaf pavement cells using FRET analysis. Dominant negative, not constitutively active, DN-rop6 could weaken the effect caused by overexpression of GDI1; because the pleiotropic phenotype of GDI1-14 plants was eliminated in the hybrid line GDI1-14 DN-rop6. GDI1 could be phosphorylated by CPK3. Three conserved Ser/Thr residues in GDI1 were determined as targeted amino acids for CPK3. Overexpression of GDI1(3D), not GDI1(3A), could rescue the abnormal growth phenotypes of gdi1-1 seedlings, demonstrating the impact of GDI1 phosphorylation in the development of Arabidopsis. In summary, these results suggest that GDI1 regulation in morphogenesis of seedlings and leaf pavement cells could be undergone through modulating the ROP signalling pathways and the phosphorylation of GDI1 by CPK3 was required for the developmental modulation in Arabidopsis.

Key words: Calcium, CPK3, GDI1, pavement cells, phosphorylation, ROP.

Introduction

The ROP (Rho of Plants) proteins are essential molecules in divergent developmental processes, such as tip growth in pollen tubes, hair elongation in roots, and pavement cell development in leaves (Fu and Yang, 2001; Fu et al., 2005; Klahre and Kost, 2006; Xu et al., 2010). Acting as versatile regulators, ROP signalling is modulated through transition between states of inactive GDP-bound and active GTP-bound while responding to diverse extracellular stimuli (Fu and Yang, 2001; Wu et al., 2001; Lavy et al., 2007). The activity of ROPs is regulated by GDP dissociation inhibitors (RhoGDIs), guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs activate Rho GTPases which are released by GDIs in cytoplasm through promoting exchange of GDP to GTP in Rho GTPases (Berken et al., 2005; Kaothien et al., 2005; Gu et al., 2006; Basu et al., 2008). GAPs control the ability of Rho GTPases to hydrolyse GTP to GDP which facilitate reinstatement of Rho GTPases in GDP-bound (Wu et al., 2000; Klahre and Kost, 2006; Hwang et al., 2008).

GDIs sequester GDP-bound soluble fractions of Rho GTPases in cytoplasm and inhibit spontaneous dissociation of GDP from Rho GTPases (Kost, 2008). Three members of RhoGDI homologues, AtRhoGDI1, AtRhoGDI2a, and AtRhoGDI2b have been identified in the Arabidopsis genome (Bischoff et al., 2000). It has been demonstrated that the AtRhoGDI1 (GDI1) may interact with ROP4 or ROP6 in vitro (Bischoff et al., 2000). The GDI1 plays essential role in root hairs growth through controlling activity of NADPH oxidase RHD2/AtrbohC in Arabidopsis (Carol et al., 2005). Coexpressing GDI1 and ROP1 in tobacco pollen tubes reduces formation of transverse actin bundles (Fu et al., 2001). In addition, deficiency in GDI2a leads to depolarization of a pollen tube growth (Hwang et al., 2010). Tobacco NtRhoGDI2 mediates recycling of NtRac5 from the flanks of the tip to the apex of a pollen tube while maintaining the polarized ROP signalling in pollen tubes (Klahre et al., 2006). Although the regulation of GDIs in the polar growth of root hairs and pollen tubes is well studied, the involvement of GDIs in other developmental processes, such as seedling development, is poorly understood.

In Arabidopsis, ROP signalling pathways are involved in an assortment of developmental aspects, including polar growth of pollen tubes, elongation of root hairs, morphogenesis of leaf pavement cells, and the response to a plant hormone signal (Lemichez et al., 2001; Baxter-Burrell et al., 2002; Fu et al., 2005; Wong et al., 2007; Yang, 2008; Xu et al., 2010). Elegant studies have demonstrated that interdigitating growth between adjacent pavement cells of Arabidopsis is precisely regulated by the plant hormone auxin. (Fu et al., 2005, 2009; Xu et al., 2010). Coordination of ROP2 and Rop-interactive CRIB motif-containing protein 4 (RIC4) promotes outgrowth of lobes through diffusely assembling cortical actin microfilaments (Fu et al., 2005), whereas collaboration of ROP6 and RIC1 suppresses lobes outgrowth through organizing microtubules (Fu et al., 2009).

The regulation of a RhoGDI by phosphorylation modification is critical for GDI function and has an impact to the binding ability of a GDI with a Rho GTPase in mammalian cells (Dransart et al., 2005). The mammalian RhoGDI1 is phosphorylated at two serine residues (Ser101 and Ser174) by PAK1 (p21-activated kinase 1) upon EGF stimulation, which promotes specific release of Rac1, indicating the key role of GDI1 phosphorylation in delivering Rac1 in mammalian cells (DerMardirossian et al., 2004). However, the dissociation of a ROP protein from the ROP-GDI complex in plant cells has not been reported, and whether GDIs perform as upstream players for ROPs in the regulation of epidermal cell growth is elusive.

This study reports that the AtRhoGDI1 (GDI1) acts as a vital regulator in development of seedlings and morphogenesis of leaf pavement cells in Arabidopsis. The interaction between GDI1 and ROP2 or ROP6 was detected in leaf pavement cells. In addition, dominant negative GDP-bound rop6 (DN-rop6) could rescue the phenotypes caused by GDI1 overexpression. Phosphorylation of GDI1 by CPK3 could be crucial for interactions of GDI1-ROP2 as well as GDI1-ROP6. The results suggest that GDI1 regulation is essential for a developing pavement cell. Phosphorylation of CPK3 on GDI1 may be a key step in the morphogenesis of pavement cells in Arabidopsis.

Materials and methods

Plant materials and growth conditions

The mutant gdi1-1 (SALK_129991) was obtained from ABRC (www.arabidopsis.org/abrc) and homozygous plants were identified with the protocol described by Alonso et al. (2003). Mutant scn1 alleles (scn1-1, scn1-2, and scn1-3) were kindly provided by Dr Liam Dolan (John Innes Centre, Norwich, UK). Surface-sterilized seeds of Arabidopsis thaliana were grown on Murashige and Skoog plates (PhytoTechnology, USA) containing 1% (w/v) sucrose and 0.8% (w/v) agar. After 2 days of stratification at 4 °C, they were transferred to a growth chamber for germination, under a 16h/8h light/dark cycle at 23 °C.

Morphometry analysis for pavement cells

The morphometry analysis for pavement cells was performed following methods described previously (Fu et al., 2005; Le et al., 2006; Sorek et al., 2011; Zhang et al., 2011). Briefly, pavement cells in one-third of the apical regions of 7-day-old cotyledons were selected for the morphometry analysis. The cotyledons were stained with propidium iodide, and then pavement cells in abaxial epidermis were imaged using confocal laser scanning microscopy (FV1000, Olympus, Japan). The lobe length of pavement cells was quantified using MetaMorph 7.5 software (Molecular Devices, USA). Over 100 cells from five independent cotyledons were measured in each independent experiment. The analytic method has been described previously (Fu et al., 2005). The area and perimeter were measured with ImageJ software (version 1.44, http://rsb.info.nih.gov/ij/). According to the protocol that was described in several reports (Fu et al., 2005; Le et al., 2006; Sorek et al., 2011; Zhang et al., 2011), the circularity of a pavement cell is defined by calculating 4π area/perimeter2 (Sorek et al., 2011). In order to quantify the number of skeleton ends of a pavement cell, the region of a pavement cell was outlined and filled with ImageJ software; then the processed image was copied and pasted as a new image. Subsequently it was analysed with the ‘plugin in’ of ‘skeleton’ embedded in ImageJ, which calculates the number of skeleton ends. Each experiment was repeated at least for three times, and the pairwise T-tests were performed for all comparisons to determine the P-values.

FRET analysis

One-week-old cotyledons from hybrid plants of GDI1-YFP, CFP-ROP2, and GDI1-YFP CFP-ROP6 (CFP, cyan fluorescent protein; GFP, green fluorescent protein; YFP, yellow fluorescent protein) were used for fluorescence resonance energy transfer (FRET) analysis. Images were acquired with confocal laser scanning microscopy. The sensitized emission method (Kraynov et al., 2000) was used for the FRET analysis. As for negative controls, pavement cells in transgenic lines expressing YFP, CFP-ROP2, or CFP-ROP6 were used. Correction factors were measured by calculating the fluorescent intensity in pavement cells of transgenic plants CFP-ROP2, CFP-ROP6, or GDI1-YFP. First, YFP fluorescent signal was acquired with excitation wavelength setting at 515nm and emission wavelength setting at 535–600nm. Then, CFP fluorescence was captured with excitation wavelength setting at 440nm and emission wavelength setting at 480nm. The FRET signal was detected with the excitation wavelength setting at 440nm and emission wavelength setting at 535–600nm. Raw FRET images and the correction factor were analysed with MetaMorph 7.5 software (Molecular Devices, USA). The bleed-through and background signals were eliminated by calculating the correction factor using MetaMorph 7.5 software; thus the corrected FRET signal was obtained. At final step, FRET efficiency was further calculated using MetaMorph 7.5 software in which the corrected FRET signal was divided by the CFP signal (Yoshizaki et al., 2003). The information about plasmids cloning used for FRET assay can be found in Supplementary Methods and Supplementary Tables S1 and S2 (available at JXB online).

Pull-down assay

The pull-down assay was performed following the method described previously (Gu et al., 2006; Wang et al., 2011) with minor modifications. Briefly, recombinant His-ROP protein (10 μg) was incubated with GST-GDI1 protein (50 μg) that was already conjugated with glutathione sepharose beads (GE Healthcare, USA) in 500 μl binding buffer (20mM Tris-HCl, pH 7.5, 150mM NaCl, 10% glycerol, 0.1% Triton X-100, 5mM MgCl2, 1mM EDTA) (Gu et al., 2006). After incubation for 1 hour, the beads were washed for five times with binding buffer to remove the unbound protein. The pulled-down protein complex was separated in 10% SDS-PAGE gel and detected by anti-His antibody (Proteintech Group, USA). For semi-in vivo pull-down assay, recombinant His-CPK3 protein was purified and conjugated to TALON beads (Clontech, USA), and then incubated with total protein extracted from 2-week-old GDI1-14 plants or 35S:GFP plants. After washing, the pulled-down protein complex was separated in 10% SDS-PAGE gel and detected with anti-GFP antibody (Proteintech Group, USA). GFP-GDI1 protein incubated with TALON beads was used as the control. The reciprocal control was using GFP protein that was pre-incubated with TALON beads and then conjugated with His-CPK3 protein. After five washes with binding buffer, the pulled-down protein complex was separated in 10% SDS-PAGE gel and detected by anti-GFP antibody (Proteintech Group, USA). Information about plasmids used in the pull-down assay is given in Supplementary Methods.

In vitro kinase assay

The recombinant protein His-CPK3 (kinase) and the substrates GST-GDI1 (full length) and GST-GDI1 fragment (Ser 2 to Asp 57) were purified from E. coli. The in vitro kinase assay was carried out following the protocol described in previous reports (Boudsocq et al., 2010; Geiger et al., 2010) with minor modifications. The reaction mixture (20 μl) was composed of 1 μg kinase protein (His-CPK3), 10 μg substrate GST-GDI1 (full length or fragment), and the reaction buffer (25mM Tris-HCl pH 7.4, 12mM MgCl2, 2mM CaCl2, 1 μM ATP, 5 μCi of [γ-32P]ATP, 1mM DTT, 1mM Na3VO4, 5mM NaF). After incubation for 30 minutes at 30 °C, the reaction was stopped by adding the sample buffer and then separated with 10% SDS-PAGE gel. The γ-32P radioactivity was detected with Typhoon 9200 phosphorimager (GE Healthcare).

Results

The involvement of GDI1 in development of seedlings and leaf epidermal cells

To investigate the role of AtRhoGDIs (GDIs) in a developmental process, this study analysed expression patterns of three Arabidopsis GDI homologues, GDI1, GDI2a, and GDI2b using reverse-transcription PCR. Results showed that GDI1 gene expression was detectable in all tested tissues of Arabidopsis. The expression of GDI2a and GDI2b was, however, predominantly showed in tissues of inflorescences and flowers (Supplementary Fig. S1A). Hence, this study attempted to explore GDI1 function in the seedling development. While analysing the T-DNA insertion mutant gdi1-1 (SALK_129991), it was found that expression level of GDI1 in mutant gdi1-1 was declined significantly (Supplementary Fig. S1B, C). The root hair growth in gdi1-1 seedlings was arrested (Supplementary Fig. S1D), which was similar to the phenotype observed in the study about scn1 mutant alleles (Carol et al., 2005). Initially it seemed that the aberrant root hair growth in gdi1-1 mutant was rescued by the YFP-tagged GDI1 fusion protein (Supplementary Fig. S1D), implying the importance of GDI1 in root hair growth. In addition, it was also noticed that transgenic plants carrying overexpressed GDI1 (with or without fluorescent protein-tagging) developed differently from those in gdi1-1 and wild-type Col-0 (WT) (Fig. 1A, Supplementary S1E). Although curly, narrowed cotyledons and true leaves were occurred in seedlings carrying overexpressed GDI1 (Fig. 1A, Supplementary S1E, F), the adult plants with overexpressed GDI1 could produce normal inflorescences and set seeds. Therefore, this study focused on exploring the influence of GDI1 in the stage of seedling development.

Fig. 1.

Fig. 1.

Open in a new tab

The phenotypes of seedling growth and cell shape in plants of gdi1 and GDI1-14. (A) A 7-day-old seedling of transgenic line GDI1-14 (GFP-GDI1) showed narrower and curly cotyledons while comparing to those in seedlings of gdi1-1 and WT (Col-0); bar, 0.5cm. (B) Enlarged mesophyll cells showed in true leaves of 3-week-old GDI1-14 seedlings; bar, 10 μm. (C) In cotyledons of 7-day-old gdi1-1 and scn1 mutant alleles the length of lobes of pavement cells (stained by propidium iodide) was shorter than that in WT, and lobes were almost disappeared in pavement cells of GDI1-14 seedlings; bar, 10 μm. (D) Geometric analysis on morphogenesis of pavement cells in plants of gdi1-1, scn1 alleles and GDI1-14; the lobe length, circularity, and number of skeleton ends were measured in the way that is modelled in upper left corner. In gdi1 mutant alleles or GDI1-14 plants, pavement cells have shorter lobes, larger circularity values, and less number of skeleton ends in comparison to WT. Data are mean ± SE of three independent experiments (n = 100 for each experiment. *P < 0.05; **P < 0.01) (this figure is available in colour at JXB online).

Several independent transgenic lines possessing higher expression level of GDI1 (Supplementary Fig. S1C) were selected for subsequent analysis. As for comparisons, transgenic plants carrying GFP-tagged GDI2b were also generated (Supplementary Fig. S1E, G). Notably, seedling growth of transgenic lines with overexpressed GDI2b showed normal (Supplementary Fig. S1E). Together, these results suggest that the diversified seedling growth in transgenic lines with overexpressed GDI1 or GDI2b might be attributed to the divergence of sequence properties in GDI1 and GDI2b proteins. In fact, the N-terminal domain of GDI1 is rather unique than those contained in GDI2b and GDI2a protein sequences (Supplementary Fig. S2). With such a unique N-terminus, GDI1 might manifest its specific regulation in the development of Arabidopsis.

To explore the internal structure of curly leaves occurred in GDI1 transgenic plants the traverse sections of leaves from GDI1-14 seedlings were investigated. Results showed that mesophyll cells in leaves of GDI1-14 seedlings were ‘swollen’ (Fig. 1B) and the shape of a leaf epidermal pavement cell was greatly altered, such as that the well-organized interlocking lobe-neck appearance (Fu et al., 2005; Xu et al., 2010) was strictly disturbed (Fig. 1C, D). The growth phenotypes of GDI1-14 plants and other transgenic lines carrying different forms of fluorescent protein-tagged GDI1 fusion constructs were further analysed. Despite which kind of fluorescent tag was fused to GDI1, overexpression of GDI1, not GDI2b, could alter morphology of epidermal pavement cells in leaves (Supplementary Fig. S1H). Consequently, attention was turned to characterize the role of GDI1 in development of leaf pavement cells.

The shape of leaf pavement cells was quantitatively analysed among plants of WT, gdi1-1 and GDI1-14 using the method of geometric analysis (Le et al., 2006; Sorek et al., 2011; Zhang et al., 2011). In order to compare the results from gdi1-1, the scn1 mutant alleles (scn1-1, scn1-2, and scn1-3) (Carol et al., 2005) were also analysed. Results indicated that average length of lobes in leaf pavement cells of gdi1-1 and scn1 was changed with comparison to that in WT (Fig. 1D). To portray the shape of a pavement cell, the circularity was used as a key parameter which was calculated based on the ratio of perimeter versus area (perimeter/area) (Fig. 1D). If a pavement cell possesses longer and narrower lobes, it has smaller value in circularity, and vice versa (Le et al., 2006; Sorek et al., 2011; Zhang et al., 2011). Using the geometric analytic method (Zhang et al., 2011), larger values of circularity for pavement cells in gdi1-1 and scn1 alleles were scored, indicating that the lobe length of pavement cells in gdi1 mutant alleles was shorter than that in WT (Fig. 1D). The lobe length of pavement cells in GDI1-14 plants was also analysed. Significant larger value of circularity was measured with GDI1-14 plants (Fig. 1D). Because the number of skeleton ends is also an important parameter to reflect the lobe numbers for a pavement cell (Sorek et al., 2011), the number of skeleton ends in plants of WT, gdi1 mutant alleles and GDI1-14 were additionally compared. The results showed that numbers of skeleton ends were quantitatively reduced in plants of gdi1 mutant alleles and GDI1-14 (Fig. 1D). Taken together, the data suggest the involvement of GDI1 in the development of leaf pavement cells. Too much or too little GDI1 may lead to aberrant networking of ROP signalling, in turn, to misregulate the morphogenesis of pavement cells.

The interaction between GDI1 and ROPs

To determine the correlation between GDI1 and ROPs (such as ROP2 and ROP6) in pavement cells, the interaction between GDI1 and ROP2 or ROP6 was examined. First, recombinant YFP signal was detected by coexpressing GDI1-YN and ROP2-YC or ROP6-YC (YC, C-terminal YFP fragment; YN, N-terminal YFP fragment) in WT mesophyll protoplasts using bimolecular fluorescence complementation assay (BiFC; Walter et al., 2004); similarly, YFP signal was detected respectively in coexpression of GDI2a-YN or GDI2b-YN and ROP2-YC or ROP6-YC (Supplementary Fig. S3A). Next, this study analysed the interaction of GDIs (GDI1, GDI2a, and GDI2b) and several ROPs (ROP2, ROP4, ROP6, ROP10, and ROP11) in WT pavement cells. Except for the coexpression of GDI2a and ROP11, and GDI2b and ROP10, the YFP signal was detectable in all examined coexpressions (Supplementary Fig. S3B). Collectively, these results demonstrate that GDI1 can interact with multiple ROPs in the pavement cell, which may function via modulation of the entire ROPs network. ROP2 and ROP6 play essential roles in pavement cell development (Fu et al., 2005, 2009; Xu et al., 2010), thus, this study attempted to examine the interaction between GDI1 and ROP2 or ROP6 in leaf pavement cells. The crossed hybrid lines GDI1-YFP CFP-ROP2 and GDI1-YFP CFP-ROP6 were analysed using the FRET assay. Not surprisingly, the FRET signal representing the interaction of GDI1 and ROP2 or ROP6 was detected in the plasma membrane and the cell cortex region of pavement cells in leaves from both hybrid lines (Fig. 2, Supplementary Fig. S3C, D). Overall, the results suggest that GDI1 regulation on ROP2 or ROP6 activity may be considered in the development of pavement cells.

Fig. 2.

Fig. 2.

Open in a new tab

Interaction between GDI1-YFP and CFP-ROP2 (upper panel) or GDI1-YFP and CFP-ROP6 (lower panel) was confirmed with FRET analysis in crossed hybrid lines; bar, 10 μm. Two enlargements show magnified views of FRET signals; bar, 2.5 μm.

To investigate the regulatory relationship between GDI1 and ROP6, the crossed hybrid lines GDI1-14 ROP6-WT, GDI1-14 CA-rop6 (G15V) and GDI1-14 DN-rop6 (T20N) were characterized. While comparing GDI1-14 ROP6-WT, normal growth of seedlings and pavement cells was observed in hybrid line GDI1-14 DN-rop6 (Fig. 3A, B). The data from quantitative analysis indicated that average length of lobes and the number of skeleton ends in pavement cells of GDI1-14 DN-rop6 seedlings were similar to those in WT seedlings (Fig. 3C), demonstrating that a rescue by DN-rop6 was incurred in hybrid line GDI1-14 DN-rop6. Interestingly, similar growth phenotype in plants of GDI1-14 and GDI1-14 CA-rop6 was notable (Fig. 3). These genetic data suggest that introducing DN-rop6 or ROP6-WT into GDI1-14 could weaken the effect caused by overexpression of GDI1; hence, the regulation of GDI1 in seedling growth and pavement cell development can be mediated by the ROP signalling.

Fig. 3.

Fig. 3.

Open in a new tab

Phenotypes of GDI1 and ROP6 hybrid lines. (A) CA-rop6 (G15V) seedling showed narrow and curly cotyledons which was similar to GDI1-14 seedling. Seedlings of hybrid lines GDI1-14 ROP6-WT and GDI1-14 DN-rop6 (T20N) showed normal growth; bar, 0.5cm. (B) Abnormal morphogenesis of pavement cells in GDI1-14 seedlings was partially reverted in hybrid lines GDI1-14 ROP6-WT and GDI1-14 DN-rop6; bar, 10 μm. (C) Geometric analysis on the pavement cells shown in (B). Shorter lobes, larger circularity values, and less number of skeleton ends were scored in GDI1-14 and GDI1-14 CA-rop6. Data are mean ± SE of three independent experiments (n = 50 for each experiment; *P < 0.05; **P < 0.01).

Impact of phosphorylation of GDI1 in seedling development

In mammalian cells, phosphorylation status of a RhoGDI is effective in the binding affinity between a RhoGDI and a Rho GTPase (Dransart et al., 2005). The mammalian RhoGDI1 can be phosphorylated by PAK1 at two serine residues (Ser101 and Ser174) upon EGF stimulation and specific release of Rac1 is then incurred, which indicates the key role of phosporylation of GDI1 in releasing Rac1 in cytosol (DerMardirossian et al., 2004). To explore the impact of phosphorylation modification of a GDI protein in seedling development, this study performed 2D-gel immunoblotting analysis for the total protein extracted from GDI1-14 plants. Several acidic forms of GDI1 protein were found (Fig. 4A). The phosphorylated peptides were enriched using the method of immobilized metal ion affinity chromatography (Chen et al., 2011). Three putative amino acids (serine 45, serine 48, and threonine 52) near the N-terminus of GDI1 were determined (Fig. 4B, Supplementary Fig. S4). To investigate the influence of phosphorylation modification to the binding affinity, the interactions of various forms of GDI1 protein and a ROP protein were analysed. The binding affinity of the non-phosphorylated form GDI1(3A) [GDI1S45AS48AT52A (3A)] or the phosphomimetic form GDI1(3D) [GDI1S45DS48DT52D (3D)] to His-tagging ROPs (ROP2, ROP6, ROP10) was examined through an in vitro pull-down assay. The results showed that GDI1(3D) possessed stronger binding affinity to His-tagging ROPs (Fig. 4C), suggesting the importance of phosphorylation status of GDI1 in modulating a ROP protein.

Fig. 4.

Fig. 4.

Open in a new tab

Analysis of GDI1 phosphorylation. (A) Acidic forms of GFP-GDI1 fusion protein (indication of arrows) were detected with 2D gel immunoblotting. (B) Schematic diagram (not scaled) indicating three phosphorylation sites (Ser45, Ser48, and Thr52) in GDI1 protein (RhoGDI domain: 36–241). (C) In vitro pull-down assay to confirm the binding affinity between phosphorylated GDI1 and ROPs. The GDI1 phosphomemetic mutant GDI(3D) (S45DS48DT52D) has stronger binding affinity to His-ROPs than the non-phosphorylated mutant GDI(3A) (S45AS48AT52A). Negative control was GST. The loading control of purified GST-tagged GDI1 is shown with Coomassie staining (Bait). His-ROPs were immunoblotted with anti-His antibody (Input). (D) In GDI1(3A) transgenic line, 5–15% seedlings displayed abnormal growth of cotyledons (three cotyledons, one cotyledon, or fused cotyledon); bar, 0.5cm. (E) Overexpressing GDI1(3A) did not affect morphogenesis of pavement cells significantly, but overexpression of GDI1 or GDI1(3D) led to severe developmental defects of pavement cells; bar, 10 μm.

To find out the impact of phosphorylation status of GDI1 in seedling development, transgenic plants carrying plasmid p35S:YFP–GDI1S45AS48AT52A (3A) and p35S:YFP–GDI1S45DS48DT52D (3D) were characterized (Supplemental Fig. S5). Cotyledon growth in some YFP-GDI1(3A) seedlings was severely affected (Fig. 4D), seedlings with three cotyledons or single cotyledon or a ‘cone-shaped’ cotyledon were observed (Fig. 4D, Supplementary Table S3). Interestingly, the shape of mature pavement cells in YFP-GDI1(3A) plants was looked normal (Fig. 4E). In sharp contrast, mature pavement cells in leaves of YFP-GDI1 and YFP-GDI1(3D) plants were wholly altered (Fig. 4E). To further confirm the influence of phosphorylation status of GDI1 in seedling development, transgenic lines of GDI1, GDI1(3D), and GDI1(3A) were introduced into gdi1-1 background respectively; thus, three transgenic lines, GDI1 (Com), 3A (Com), and 3D (Com), were analysed. The development of pavement cells and root hairs in plants of GDI1 (Com) and 3D(Com) was recovered to normal growth; however, the recovery was not observed in 3A(Com) plants (Supplementary Fig. S5). Taken together, these results demonstrate a significant impact of phosphorylation status of GDI1 in development of seedlings and pavement cells in Arabidopsis.

Phosphorylation of GDI1 by CPK3

Because the consensus sequence R-X-X-S that has been reported as the target domain for calcium-dependent protein kinases (CPKs) (Cheng et al., 2002) is contained in GDI1 protein (Fig. 4B), this study examined the possible phosphorylation modification of CPKs to GDI1. Total 34 CDPKs were determined in Arabidopsis (Cheng et al., 2002), among which CPK3, CPK9, CPK10, CPK28, and CPK32 have been reported to possess abundant expression levels in leaves (Boudsocq et al., 2010). This study attested the phosphorylation activities of CPKs on GDI1 and found out that CPK3 could phosphorylate the full length and the peptide fragment of GDI1 protein (Ser 2 to Asp 57) in vitro (Fig. 5A). Not surprisingly, a declined phosphorylation level was observed in GDI1(3A) (Fig. 5A). Furthermore, this study investigated the interaction between GDI1 and CPK3 and demonstrated the existence of interaction between GDI1 and CPK3 in vitro and in vivo (Figs. 5B, 5C). To substantiate the involvement of calcium signalling in developing epidermal cells, this study examined the effect of manipulating Ca2+ release from intracellular calcium stores in the treatment of 2-aminoethyl diphenylborinate (2-APB), an inhibitor of intracellular calcium stores (Engstrom et al., 2002). As results, the average length of lobes of pavement cells in treated WT seedlings was reduced whereas the circularity of pavement cells in treated WT seedlings was increased (Supplementary Fig. S6). Additionally, seedlings were treated with N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride (W-7), the calmodulin antagonist that has been used for studying CDPKs activities in several reports (Romeis et al., 2000; Kaplan et al., 2006). Notably, the shape of pavement cells in WT cotyledons was obviously altered with treatment (Supplementary Fig. S6). Collectively, these results demonstrate the significance of calcium signalling in the development of pavement cells.

Fig. 5.

Fig. 5.

Open in a new tab

GDI1 phosphorylated by CPK3 in vitro. (A) CPK3 phosphorylates GDI1 in vitro. Purified His-CPK3 was incubated with GST-GDI1, GST-GDI1S45AS48AT52A (3A), Histone (positive control), and GST-GDI1 fragment (Ser 2 to Asp 57) in the presence of [γ-32P]ATP respectively. The [γ-32P]ATP labelled protein bands are indicated with arrows. (B) Interaction between CPK3 and GDI1 was detected in the BiFC assay (right): YC, C-terminal YFP fragment; YN, N-terminal YFP fragment. Protoplasts expressing pCPK3-YC and vector p35S:YN was used for negative control (left); bar, 5 μm. (C) Semi-in vivo pull-down assay to confirm the interaction between GDI1 and CPK3. His-CPK3-conjugated TALON beads were incubated with total proteins purified from 2-week-old transgenic plants of 35S:GFP (control line) and GDI1-14. The pulled-down protein complex was detected with anti-GFP antibody. GFP-GDI1 protein incubated with non-conjugated TALON beads (beads+GFP-GDI1) was used for the control. Reciprocally GFP protein incubated with His-CPK3 (His-CPK3+GFP) was also used for the control. The loading control is shown with Coomassie staining (Bait). The input total proteins extracted from 35S:GFP and GDI1-14 transgenic plants were immunoblotted with anti-GFP antibody.

Discussion

Diverse regulations of GDI1 to a ROP might exist in Arabidopsis

RhoGDIs are initially depicted as negative regulators and capable of inhibiting the dissociation of GDP from Rho GTPases in eukaryotic cells (Dransart et al., 2005), which extract the Rho GTPases from plasma membrane to maintain the inactivated Rho GTPases in cytoplasm. Additionally, RhoGDIs can also inhibit the hydrolytic activities of Rho GTPases in mammalian cells (Boulter et al., 2010; Garcia-Mata et al., 2011). RhoGDI1 in mammalian cells is not only able to control stability and homeostasis of multiple Rho GTPases but also acts as a chaperon to facilitate the translocation of Rho GTPases from cytoplasm to the plasma membrane (Boulter et al., 2010). The regulation of RhoGDIs in plant cells, however, is poorly understood. The current study discusses the role of AtRhoGDI1 (GDI1) in modulating the development of Arabidopsis seedlings. Overexpression of GDI1 could trigger abnormal growth of seedlings and irregular morphology of leaf epidermal pavement cells, which were similar to those shown in CA-rop6 transgenic plants. Interestingly, the abnormality of GDI1 transgenic plants can be rescued by DN-rop6, not CA-rop6 (Fig. 3). It is considered that DN-rop6 might weaken the effect caused by overexpression of GDI1 through binding to excessive amount of GDI1 protein in pavement cells, thereby balancing the interference of overexpressed GDI1. In a similar fashion, introducing ROP6-WT into GDI1 background could also rescue the effect of overexpressed GDI1. These results suggest the complexity of GDI1 regulation during seedling development. Concerning the shorter lobes shown in abnormally developed pavement cells, a severe phenotype was produced in pavement cells of plants carrying overexpressed GDI1 (Fig. 1). Shorter lobes of pavement cells in plants with positively or negatively modulated ROP activity have been reported previously (Fu et al., 2002). For example, CA-rop2 and DN-rop2 play opposite roles in regulating cytoskeleton arrangement; however, expression of these two forms of ROP2 leads to shorter lobes in pavement cells (Fu et al., 2005). The current study hypothesizes that, in addition to playing as the GDP dissociation inhibitor for ROPs, AtRhoGDI1 might take part in stabilizing and distributing ROPs in pavement cells. The results of the interactions of GDI1-ROP2 and GDI1-ROP6 at the cell cortex near the plasma membrane region (Fig. 2) implicate the regulation of GDI1 on the ROP function, which is in agreement with the theory that ROP2 acts mostly at the plasma membrane of lobe region and that ROP6 serves preferentially at the plasma membrane of indentation zone in leaf epidermal pavement cells in Arabidopsis (Fu et al., 2002, 2005; Xu et al., 2010). Possibly, during development of leaf pavement cells AtRhoGDI1 could also perform as a chaperon to transport ROP2 to its destination (such as to the cell cortex near lobes) while promoting dissociation of ROP6 from the plasma membrane of the lobe region. Thus, AtRhoGDI1 might possess multiple functions as those that are demonstrated in mammalian cells (Garcia-Mata et al., 2011). In addition, AtRhoGDI1 can regulate multiple ROPs in pavement cells. As tested in this study, except for interacting with ROP2 and ROP6, GDI1 can also interact with ROP4, ROP10, and ROP11. Future studies underlying regulation of the GDI1 in distribution and activity of ROPs in plant cells will provide further insight.

Phosphorylation of GDI1 by CPK3 implicates convergence of multiple signalling pathways

The phosphorylation state of RhoGDIs by the protein kinase is a regulatory mechanism for dissociation of Rho GTPases in mammalian cells. The p21-activated kinase 1 (PAK1), a downstream effector of Rac1 and Cdc42, is demonstrated to phosphorylate RhoGDI1 at two amino acids (Ser101 and Ser174) (DerMardirossian et al., 2004). In plant cells, little is known about post-translational modifications of GDIs. This study determined that, CPK3, a calcium-dependent protein kinase, is possible to phosphorylate AtRhoGDI1. Three amino acids, Ser45, Ser48, and Thr52, conserved in GDI1 protein, are key target sites for CPK3. The phosphorylation status of GDI1 influences its binding ability to ROPs. Although the developed pavement cells are looked normal in GDI1(3A) seedlings, the growth of cotyledons can be severely altered (Fig. 4), which is similar to phenotypes observed in auxin-related mutants (Benková et al., 2003; Jaillais et al., 2007). For example, three cotyledons are observed in mutant vps29 (vacuolar protein sorting 29) due to disturbed PIN1 polarity (Jaillais et al., 2007). NtRac1 can stimulate expressions of auxin-responsive genes and mediate 26S proteasome-dependent proteolysis of AUX/IAAs proteins (Tao et al., 2002, 2005). In leaf pavement cells, the localization of PIN1 can be affected by the ROP signalling, in turn, the auxin response is subsequently modified (Xu et al., 2010). The current characterization of the transgenic lines GDI1(3A) and GDI1(3D) may imply the importance of phosphorylation status of GDI1 in the auxin-regulated developmental processes. A future study to determine the role of GDI1 in auxin polar transport and/or auxin signal transduction may widen the knowledge on this point.

CPK3 is activated in response to salt stress, and it is functional in the ABA-regulated ion channels in guard cells (Mori et al., 2006; Mehlmer et al., 2010). The results of this study suggest a potential role of CPK3 involving in ROP-mediated developmental processes. As an important calcium sensor in plant cells, CPKs have been demonstrated to play diverse roles in modulating polar growth, including root hair growth and pollen tube elongation (Ivashuta et al., 2005; Yoon et al., 2006; Myers et al., 2009). Here it is shown that calcium-mediated signalling in morphogenesis of an epidermal cell may require CPK3-phosphorylated GDI1. Concerning the functional redundancy (Cheng et al., 2002), the functions of other CPKs in phosphorylation of GDI1 need to be clarified.

Overall, this study provides evidence of a role of GDI1 regulation in the development of seedlings and pavement cells in Arabidopsis. The CPK3 phosphorylation of GDI1 may confer a fresh insight to further understand the convergence of extracellular signalling and intracellular ROP signalling pathways, including calcium-dependent modulations.

Supplementary material

Supplementary data are available at JXB online.

Supplementary Fig. S1. Characterization of expression patterns of GDI genes and phenotypes of GDI1 transgenic plants.

Supplementary Fig. S2. Sequence comparisons of GDIs homologues.

Supplementary Fig. S3. Analysis of interactions between GDIs and ROPs.

Supplementary Fig. S4. Determination of phosphorylation sites of GDI1 protein.

Supplementary Fig. S5. Characterizations of GDI1 transgenic lines carrying mutated phosphorylation sites.

Supplementary Fig. S6. Analysis on effects of calcium level and CPK activity in pavement cell morphogenesis.

Supplementary Table S1. Plasmids used in this study.

Supplementary Table S2. Primer sequences for gdi1-1 homozygote (HM) identification, plasmids construction, and reverse-transcription PCR.

Supplementary Table S3. Quantitative analysis on cotyledon phenotypes in YFP-GDI1(3A) transgenic lines.

Supplementary methods.

Supplementary Data
supp_64_11_3327__index.html (1,000B, html)

Acknowledgements

The authors thank the Wu lab members for technique help and helpful comments on the manuscripts. They are grateful to Dr Liam Dolan (John Innes Centre, Norwich, UK) for providing scn1-1, scn1-2, and scn1-3 seeds, Dr Nam-Hai Chua (Rockefeller University, USA) for providing the plasmid pBA002, and Syngenta for providing the Friendly Laboratories Scholarship to Yuxuan Wu. This work was supported by grants to Yan Wu from the National Natural Science Foundation of China (31270333, 90817013) and the Ministry of Science and Technology of China (2013CB126900).

Glossary

Abbreviations:

GAP

GTPase-activating protein

GDI

GDP dissociation inhibitor

GEF

guanine nucleotide exchange factor.

References

  1. Alonso JM, Stepanova AN, Leisse TJ, et al. 2003. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657 [DOI] [PubMed] [Google Scholar]
  2. Basu D, Le J, Zakharova T, Mallery EL, Szymanski DB. 2008. A SPIKE1 signaling complex controls actin-dependent cell morphogenesis through the heteromeric WAVE and ARP2/3 complexes. Proceedings of the National Academy of Sciences, USA 105, 4044–4049 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baxter-Burrell A, Yang ZB, Springer PS, Bailey-Serres J. 2002. RopGAP4-dependent Rop GTPase rheostat control of Arabidopsis oxygen deprivation tolerance. Science 296, 2026–2028 [DOI] [PubMed] [Google Scholar]
  4. Benková E, Michniewicz M, Sauer M, Teichmann T, Seifertová D, Jürgens G, Friml J. 2003. Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 115, 591–602 [DOI] [PubMed] [Google Scholar]
  5. Berken A, Thomas C, Wittinghofer A. 2005. A new family of RhoGEFs activates the Rop molecular switch in plants. Nature 436, 1176–1180 [DOI] [PubMed] [Google Scholar]
  6. Bischoff F, Vahlkamp L, Molendijk A, Palme K. 2000. Localization of AtROP4 and AtROP6 and interaction with the guanine nucleotide dissociation inhibitor AtRhoGDI1 from Arabidopsis. Plant Molecular Biology 42, 515–530 [DOI] [PubMed] [Google Scholar]
  7. Boudsocq M, Willmann MR, McCormack M, Lee H, Shan LB, He P, Bush J, Cheng SH, Sheen J. 2010. Differential innate immune signalling via Ca2+ sensor protein kinases. Nature 464, 418–422 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Boulter E, Garcia-Mata R, Guilluy C, Dubash A, Rossi G, Brennwald PJ, Burridge K. 2010. Regulation of Rho GTPase crosstalk, degradation and activity by RhoGDI1. Nature Cell Biology 12, 477–483 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carol RJ, Takeda S, Linstead P, Durrant MC, Kakesova H, Derbyshire P, Drea S, Zarsky V, Dolan L. 2005. A RhoGDP dissociation inhibitor spatially regulates growth in root hair cells. Nature 438, 1013–1016 [DOI] [PubMed] [Google Scholar]
  10. Chen X, Wu D, Zhao Y, Wong BHC, Guo L. 2011. Increasing phosphoproteome coverage and identification of phosphorylation motifs through combination of different HPLC fractionation methods. Journal of Chromatography B 879, 25–34 [DOI] [PubMed] [Google Scholar]
  11. Cheng SH, Willmann MR, Chen HC, Sheen J. 2002. Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family. Plant Physiology 129, 469–485 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. DerMardirossian C, Schnelzer A, Bokoch GM. 2004. Phosphorylation of RhoGDI by Pak1 mediates dissociation of Rac GTPase. Molecular Cell 15, 117–127 [DOI] [PubMed] [Google Scholar]
  13. Dransart E, Olofsson B, Cherfils J. 2005. RhoGDIs revisited: novel roles in Rho regulation. Traffic 6, 957–966 [DOI] [PubMed] [Google Scholar]
  14. Engstrom EM, Ehrhardt DW, Mitra RM, Long SR. 2002. Pharmacological analysis of nod factor-induced calcium spiking in Medicago truncatula. Evidence for the requirement of type IIA calcium pumps and phosphoinositide signaling. Plant Physiology 128, 1390–1401 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fu Y, Gu Y, Zheng ZL, Wasteneys G, Yang ZB. 2005. Arabidopsis interdigitating cell growth requires two antagonistic pathways with opposing action on cell morphogenesis. Cell 120, 687–700 [DOI] [PubMed] [Google Scholar]
  16. Fu Y, Li H, Yang ZB. 2002. The ROP2 GTPase controls the formation of cortical fine F-actin and the early phase of directional cell expansion during Arabidopsis organogenesis. The Plant Cell 14, 777–794 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fu Y, Wu G, Yang ZB. 2001. Rop GTPase-dependent dynamics of tip-localized F-actin controls tip growth in pollen tubes. Journal of Cell Biology 152, 1019–1032 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fu Y, Xu TD, Zhu L, Wen MZ, Yang ZB. 2009. A ROP GTPase signaling pathway controls cortical microtubule ordering and cell expansion in Arabidopsis. Current Biology 19, 1827–1832 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fu Y, Yang ZB. 2001. Rop GTPase: a master switch of cell polarity development in plants. Trends in Plant Science 6, 545–547 [DOI] [PubMed] [Google Scholar]
  20. Garcia-Mata R, Boulter E, Burridge K. 2011. The ‘invisible hand’: regulation of RHO GTPases by RHOGDIs. Nature Reviews Molecular Cell Biology 12, 493–504 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Geiger D, Scherzer S, Mumm P, et al. 2010. Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ affinities. Proceedings of the National Academy of Sciences, USA 107, 8023–8028 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gu Y, Li SD, Lord EM, Yang ZB. 2006. Members of a novel class of Arabidopsis Rho guanine nucleotide exchange factors control rho GTPase-dependent polar growth. The Plant Cell 18, 366–381 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hwang JU, Vernoud V, Szumlanski A, Nielsen E, Yang ZB. 2008. A tip-localized RhoGAP controls cell polarity by globally inhibiting Rho GTPase at the cell apex. Current Biology 18, 1907–1916 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hwang JU, Wu G, Yan A, Lee YJ, Grierson CS, Yang ZB. 2010. Pollen-tube tip growth requires a balance of lateral propagation and global inhibition of Rho-family GTPase activity. Journal of Cell Science 123, 340–350 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ivashuta S, Liu JY, Liu JQ, Lohar DP, Haridas S, Bucciarelli B, VandenBosch KA, Vance CP, Harrison MJ, Gantt JS. 2005. RNA interference identifies a calcium-dependent protein kinase involved in Medicago truncatula root development. The Plant Cell 17, 2911–2921 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jaillais Y, Santambrogio M, Rozier F, Fobis-Loisy I, Miège C, Gaude T. 2007. The retromer protein VPS29 links cell polarity and organ initiation in plants. Cell 130, 1057–1070 [DOI] [PubMed] [Google Scholar]
  27. Kaothien P, Ok SH, Shuai B, Wengier D, Cotter R, Kelley D, Kiriakopolos S, Muschietti J, McCormick S. 2005. Kinase partner protein interacts with the LePRK1 and LePRK2 receptor kinases and plays a role in polarized pollen tube growth. The Plant Journal 42, 492–503 [DOI] [PubMed] [Google Scholar]
  28. Kaplan B, Davydov O, Knight H, Galon Y, Knight MR, Fluhr R, Fromm H. 2006. Rapid transcriptome changes induced by cytosolic Ca2+ transients reveal ABRE-related sequences as Ca2+-responsive cis elements in Arabidopsis. The Plant Cell 18, 2733–2748 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Klahre U, Becker C, Schmitt AC, Kost B. 2006. Nt-RhoGDI2 regulates Rac/Rop signaling and polar cell growth in tobacco pollen tubes. The Plant Journal 46, 1018–1031 [DOI] [PubMed] [Google Scholar]
  30. Klahre U, Kost B. 2006. Tobacco RhoGTPase ACTIVATING PROTEIN1 spatially restricts signaling of RAC/Rop to the apex of pollen tubes. The Plant Cell 18, 3033–3046 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kost B. 2008. Spatial control of Rho (Rac-Rop) signaling in tip-growing plant cells. Trends in Cell Biology 18, 119–127 [DOI] [PubMed] [Google Scholar]
  32. Kraynov VS, Chamberlain C, Bokoch GM, Schwartz MA, Slabaugh S, Hahn KM. 2000. Localized Rac activation dynamics visualized in living cells. Science 290, 333–337 [DOI] [PubMed] [Google Scholar]
  33. Lavy M, Bloch D, Hazak O, Gutman I, Poraty L, Sorek N, Sternberg H, Yalovsky S. 2007. A novel ROP/RAC effector links cell polarity, root-meristern maintenance, and vesicle trafficking. Current Biology 17, 947–952 [DOI] [PubMed] [Google Scholar]
  34. Le J, Mallery EL, Zhang CH, Brankle S, Szymanski DB. 2006. Arabidopsis BRICK1/HSPC300 is an essential WAVE-complex subunit that selectively stabilizes the Arp2/3 activator SCAR2. Current Biology 16, 895–901 [DOI] [PubMed] [Google Scholar]
  35. Lemichez E, Wu Y, Sanchez JP, Mettouchi A, Mathur J, Chua NH. 2001. Inactivation of AtRac1 by abscisic acid is essential for stomatal closure. Genes and Development 15, 1808–1816 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mehlmer N, Wurzinger B, Stael S, Hofmann-Rodrigues D, Csaszar E, Pfister B, Bayer R, Teige M. 2010. The Ca2+-dependent protein kinase CPK3 is required for MAPK-independent salt-stress acclimation in Arabidopsis. The Plant Journal 63, 484–498 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mori IC, Murata Y, Yang YZ, et al. 2006. CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca2+-permeable channels and stomatal closure. PLoS Biology 4, 1749–1762 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Myers C, Romanowsky SM, Barron YD, Garg S, Azuse CL, Curran A, Davis RM, Hatton J, Harmon AC, Harper JF. 2009. Calcium-dependent protein kinases regulate polarized tip growth in pollen tubes. The Plant Journal 59, 528–539 [DOI] [PubMed] [Google Scholar]
  39. Romeis T, Piedras P, Jones JDG. 2000. Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defense response. The Plant Cell 12, 803–815 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sorek N, Gutman O, Bar E, et al. 2011. Differential effects of prenylation and s-acylation on type I and II ROPS membrane interaction and function. Plant Physiology 155, 706–720 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tao LZ, Cheung AY, Nibau C, Wu HM. 2005. RAC GTPases in tobacco and Arabidopsis mediate auxin-induced formation of proteolytically active nuclear protein bodies that contain AUX/IAA proteins. The Plant Cell 17, 2369–2383 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tao LZ, Cheung AY, Wu HM. 2002. Plant Rac-like GTPases are activated by auxin and mediate auxin-responsive gene expression. The Plant Cell 14, 2745–2760 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Walter M, Chaban C, Schutze K, et al. 2004. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. The Plant Journal 40, 428–438 [DOI] [PubMed] [Google Scholar]
  44. Wang YP, Li L, Ye TT, Zhao SJ, Liu Z, Feng YQ, Wu Y. 2011. Cytokinin antagonizes ABA suppression to seed germination of Arabidopsis by downregulating ABI5 expression. The Plant Journal 68, 249–261 [DOI] [PubMed] [Google Scholar]
  45. Wong HL, Pinontoan R, Hayashi K, et al. 2007. Regulation of rice NADPH oxidase by binding of Rac GTPase to its N-terminal extension. The Plant Cell 19, 4022–4034 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wu G, Gu Y, Li SD, Yang ZB. 2001. A genome-wide analysis of Arabidopsis Rop-interactive CRIB motif-containing proteins that act as Rop GTPase targets. The Plant Cell 13, 2841–2856 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wu G, Li H, Yang ZB. 2000. Arabidopsis RopGAPs are a novel family of Rho GTPase-activating proteins that require the Cdc42/Rac-interactive binding motif for Rop-specific GTPase stimulation. Plant Physiology 124, 1625–1636 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Xu TD, Wen MZ, Nagawa S, et al. 2010. Cell surface- and Rho GTPase-based auxin signaling controls cellular interdigitation in ArabidopsisCell143, 99–110 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yang ZB. 2008. Cell polarity signaling in Arabidopsis. Annual Review of Cell and Developmental Biology 24, 551–575 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Yoon GM, Dowd PE, Gilroy S, McCubbin AG. 2006. Calcium-dependent protein kinase isoforms in Petunia have distinct functions in pollen tube growth, including regulating polarity. The Plant Cell 18, 867–878 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yoshizaki H, Ohba Y, Kurokawa K, Itoh RE, Nakamura T, Mochizuki N, Nagashima K, Matsuda M. 2003. Activity of Rho-family GTPases during cell division as visualized with FRET-based probes. Journal of Cell Biology 162, 223–232 [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zhang C, Halsey LE, Szymanski DB. 2011. The development and geometry of shape change in Arabidopsis thaliana cotyledon pavement cells. BMC Plant Biology 11, 27 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data
supp_64_11_3327__index.html (1,000B, html)
supp_ert171_jexbot096487_file001.pdf (995KB, pdf)

Articles from Journal of Experimental Botany are provided here courtesy of Oxford University Press

RESOURCES