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First published online March 16, 2005; 10.1105/tpc.104.027474 © 2005 American Society of Plant Biologists
Leucine-Rich Repeat Receptor-Like Kinase1 Is a Key Membrane-Bound Regulator of Abscisic Acid Early Signaling in Arabidopsis
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ABSTRACT |
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INTRODUCTION |
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; Finkelstein et al., 2002). Many genes that play important roles in the ABA signaling pathway have been identified by molecular and genetic approaches. They include genes for transcription factors (ABI3, ABI4, and ABI5), protein phosphatases (ABI1 and ABI2), protein kinases, a farnesyl transferase (ERA1), a phospholipase D (PLD 
), an SnRK2 protein kinase (SRK2E/OST1), a G protein, and small GTPases (Li et al., 2000; Lemichez et al., 2001; McCourt, 2001; Sang et al., 2001; Finkelstein et al., 2002; Guo et al., 2002; Mustilli et al., 2002; Yoshida et al., 2002; Zheng et al., 2002; Coursol et al., 2003). Recently, proteins involved in posttranscriptional regulation and protein turnover were shown to modify ABA signaling pathways (Lu and Fedoroff, 2000; Hugouvieux et al., 2001; Xiong et al., 2001). However, the important signaling molecules or the principal proteins involved in the ABA perception machinery have not been identified genetically. Few ABA binding proteins have been identified biochemically.
Previous studies thus far suggest that protein phosphorylation/dephosphorylation is an important regulator of ABA signaling. The ABA signal is perceived at both cytoplasmic and extracellular sites (Finkelstein et al., 2002), and signaling events should occur at the plasma membrane for extracellular ABA perception (Yamazaki et al., 2003). ABA microinjected into the cytoplasm of guard cells was able to trigger stomatal closure and inhibit inward K+ currents (Allan et al., 1994; Schwartz et al., 1994). Extracellularly applied ABA–BSA conjugate, which cannot enter the cell, was able to trigger RAB18 or Em gene expression, stimulate membrane depolarization, and activate ion channels (Schultz and Quatrano, 1997; Jeannette et al., 1999). Furthermore, ABA binding proteins have been detected in the microsome fraction of Arabidopsis by the use of ABA–protein conjugates as affinity probes (Pedron et al., 1998). These findings show that the plasma membrane is one of the most important sites for the perception of ABA in Arabidopsis.
Leu-rich repeat receptor-like protein kinases (LRR-RLKs) that contain an extracellular LRR and a Ser/Thr kinase domain and are localized at the plasma membrane are important in the transduction of various plant environmental and developmental signals (Tichtinsky et al., 2003). Many LRR-RLKs are encoded in the plant genome (Shiu and Bleecker, 2001), but most of their functions are unknown, with the exception of several well-characterized proteins, such as CLAVATA1 (Clark et al., 1997), a brassinosteroid (BR) receptor, BR-insensitive1 (BRI1) (Wang et al., 2001a), a systemin receptor (Scheer and Ryan, 2002), a flagellin receptor, FLS2 (Gomez-Gomez et al., 2001), and a phytosulfokine receptor (Matsubayashi et al., 2002). These reports show that the LRR domain alone, or the protein complex, functions as a receptor of both peptide and nonpeptide hormones.
RPK1 is an LRR-RLK isolated from Arabidopsis thaliana, and expression of the gene is induced by ABA, dehydration, high salt, and low temperature (Hong et al., 1997). To determine the function of RPK1 in ABA perception in plants, we analyzed the expression pattern of RPK1 at the cellular level to show the localization of RPK1 on the plasma membrane. We also analyzed RPK1 knockout mutants and antisense-RPK1 transgenic lines. The loss of function of RPK1 showed ABA insensitivity in seed germination, plant growth, stomatal closure, and gene expression in Arabidopsis. We present several lines of evidence for an important role of RPK1 in ABA signal transduction. On the basis of our results, we discuss the function of RPK1 in ABA perception and signal transduction.
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). To determine the role of RPK1 in ABA signaling, we analyzed the effects of various plant hormone treatments, including ABA, on the expression of RPK1 (Figure 1A). RPK1 was expressed at low levels, even under control conditions, and its mRNA accumulation was specifically upregulated by ABA but not by other plant hormones, namely, ethephon, salicylic acid (SA), methyl jasmonate (MeJA), gibberellic acid (GA3), cytokinin (6-benzylaminopurine [BA]), and auxin (2,4-D). RPK1 protein was detected in untreated plants by protein gel blot analysis using anti-RPK1 polyclonal antibody (Figure 1B). Specific upregulation of RPK1 by ABA was also shown at the protein level in mature Columbia ecotype plants and T87 suspension-cultured cells (Figure 1B). Figure 1C shows the expression pattern of the RPK1 promoter–ß-glucuronidase (GUS) fusion gene in transgenic Arabidopsis. GUS activity was increased by ABA treatment (Figure 1C, 1), especially in the cotyledons and vascular tissues; a low level of activity was found in untreated transgenic plants (Figure 1C, 2). Strong activity was also found in embryos of mature seeds (Figure 1C, 3). These expression patterns indicate that expression of the RPK1 gene is activated specifically by endogenous as well as exogenous ABA.
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ABA Insensitivity of RPK1 Knockouts and Antisense-RPK1 Transgenic Plants
To analyze the function of RPK1 in ABA signaling in plant cells, we screened T-DNA–tagged Arabidopsis Wassilewskija (Ws) lines from the Arabidopsis Knockout Facility collection at Wisconsin University and obtained two RPK1 knockout mutants, rpk1-1 and rpk1-2, after PCR screening (Figure 2A). T-DNAs were inserted 504 and 989 bp downstream of the translational start site of the RPK1 gene (Figure 2B) in rpk1-1 and rpk1-2, respectively, and were confirmed by the sequences of the PCR products. RPK1 expression was repressed in rpk1-1 and rpk1-2 homozygous lines at both transcription and translation levels (Figure 2A). These plants do not show any morphological alterations under normal growth conditions (data not shown). We examined the ABA sensitivities of the RPK1 knockouts plants. The germination rates of the RPK1 knockouts were higher than those of wild-type plants on both ABA-containing and ABA-free media (Figure 2C). Furthermore, overexpression of RPK1 in rpk1-1 (Figure 2C, 35S:RPK1rpk1-1) was able to complement the ABA insensitivity. Figure 2D shows the germination rates of the wild-type and RPK1 knockout plants on ABA-containing media at various concentrations of ABA. The slightly higher germination rate without ABA might indicate insensitivity to internal ABA. Thus, the RPK1 knockout plants showed an ABA-insensitive phenotype during germination. The negative effect of ABA on root growth was also reduced in the RPK1 knockouts (Figure 2E).
We then generated antisense-RPK1 transgenic plants in which RPK1 mRNA accumulation was suppressed under the 35S promoter of Cauliflower mosaic virus. Antisense-RPK1 plants also showed higher germination rates with or without ABA than did controls (Figure 3C). RPK1 suppression in the antisense-RPK1 Arabidopsis lines was clearer in lines L1 and L2 than in L3 and vector control (VC) plants (Figure 3A). The antisense-RPK1 L1 and L2 plants showed a stronger phenotype than the L3 plants (Figure 3C). These results indicate that RPK1 repression resulted in ABA insensitivity in Arabidopsis.
We also generated antisense-RPK1 transgenic T87 suspension cell lines whose RPK1 protein levels were clearly decreased on protein gel blot analysis (Figure 3B). Suspension culture cells contain specific cell regulation mechanisms that maintain proliferation processes. We analyzed the negative effect of ABA on Arabidopsis cell growth (Figure 3D). In ABA-containing media, VC cells grew poorly and showed arrested cell reproduction; the antisense-RPK1 cell lines continued growing. These antisense transgenic cells remained green (data not shown), whereas VC cells turned yellow in ABA-containing media. This result indicates that RPK1 has an elemental role in cell proliferation that is controlled by ABA. These ABA-insensitive phenotypes of the RPK1 knockouts and antisense transgenic plants suggest that RPK1 is an important positive regulator of the ABA signal transduction pathway of Arabidopsis.
ABA Insensitivity of the rpk1 Mutants in Plant Growth and Stomatal Closure of Guard Cells
We further analyzed the ABA insensitivities of RPK1 knockouts (Figure 4). The growth rate of wild-type Arabidopsis was severely inhibited by exogenously applied ABA. When knockout plants were grown on seed germination media (GM) agar plates containing ABA, growth retardation by ABA was suppressed markedly in the rpk1 knockout plants compared with the wild-type plants (Figure 4A). ABA promotes stomatal closure in guard cells to avoid water loss during drought stress. When control plants were treated with 5 µM ABA for 2 h, most of the stomata were closed. In the RPK1 knockout plants under the same condition, the stomata remained open (Figures 4B and 4C). These results indicate that loss of function of RPK1 causes ABA insensitivity also during plant growth and stomatal closure, suggesting that RPK1 is a positive regulator of the ABA response and regulates diverse ABA responses in development and stress responses.
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) using mRNA prepared from ABA-treated antisense-RPK1 plants and T87 cells was performed and results are shown in Supplemental Table 1 online. The expression patterns of several downregulated genes in antisense-RPK1 transgenic plants were confirmed by RNA gel blot analysis (Figure 5A). Expression levels of other well-known genes regulated by ABA, such as ADH1 (MacNicol and Jacobsen, 2001), RD22, MYC2, AREB1, and several ERD genes (Shinozaki and Yamaguchi-Shinozaki, 2000), also decreased in antisense-RPK1 plants. RNA gel blot analysis revealed that the expression levels of most ABA-inducible genes were decreased in the rpk1-1 mutant (Figure 5A). Expression of several genes that were downregulated in antisense-RPK1 plants was not affected in the rpk1-1 mutant. This different expression may be due to differences in the ecotypes of Arabidopsis and/or to the existence of RPK1 homologs in the Arabidopsis genome, whose expressions might be suppressed in antisense-RPK1 transgenic plants.
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), were also suppressed.
We then attempted to identify the RPK1-regulated genes in the rpk1-2 knockout mutant during seedling development using an Agilent Arabidopsis 2 Oligo Microarray (Agilent Technologies, Palo Alto, CA), which covers >21,000 Arabidopsis genes. Total RNA samples were prepared from ABA-treated rpk1-2 and wild-type plants and used for microarray analysis. We performed two experiments for each RNA sample using different labels, cy3 or cy5. The downregulated genes with ratios of <0.5 are summarized in Table 1 and in Supplemental Tables 2 and 3 online. Among these downregulated genes in rpk1-2, we found the genes for 2S storage protein, 12S storage protein, type 2 peroxiredoxin, and oleosin, which are regulated by ABI3 in the embryo (Kroj et al., 2003). Other seed storage proteins, such as pEARLI1 and legumin proteins, were also included in the downregulated genes. Approximately 44% of the downregulated genes were ABA inducible, as confirmed by the Agilent Arabidopsis 2 Oligo Microarray with 3-week-old Arabidopsis plants (K. Maruyama, unpublished data).
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; Goda et al., 2004). The results suggest that the BR signaling pathway is repressed in the RL-BK dwarf transgenic plants. On the other hand, we found that many upregulated genes in the RL-BK overexpressors with a ratio of >3.0 (Table 3) contain ABA-inducible genes and seed storage protein genes. The RL-BK transgenic plants showed delayed seed germination on ABA-containing media, suggesting increased sensitivity to ABA. This result suggested that the RL-BK physiological response to ABA is consistent with the microarray data (Figure 7D).
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DISCUSSION |
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To clarify the RPK1 function during the cell growth of cultured cells, we constructed antisense-RPK1 transgenic suspension culture cells and analyzed their ABA sensitivity during cell proliferation. The control T87 cells showed arrested cell reproduction in the presence of ABA, whereas the antisense-RPK1 cell lines continued growing in the presence of ABA (Figure 3). ABA was found to prevent DNA replication, keeping the cells in the G1 stage, when it was applied just before the G1/S transition of tobacco (Nicotiana tabacum) BY-2 cells (Swiatek et al., 2002). This ABA-mediated inhibition of cell cycle progression may be repressed in the antisense-RPK1 transgenic culture cells, suggesting the key role of RPK1 in ABA signaling in cultured cells.
The microarray analysis of the RPK1 mutant and antisense-RPK1 transgenic plants showed reduced expression levels of various ABA-inducible genes (Figure 5, Table 1; see Supplemental Table 1 online). Microarray analyses using ABA-treated whole plants (Seki et al., 2002c), the abi1-1 mutant (Hoth et al., 2002), and guard cells (Leonhardt et al., 2004) have revealed a diversity of ABA-regulated genes. We found many such ABA-regulated genes in the results of our microarray analysis, indicating that RPK1 positively regulates many ABA-inducible genes that are controlled through various ABA-dependent signal transduction pathways. Previous genetic studies have shown that various negative regulators, such as ABI1, ABI2, ERA1, ROP10, AtRac1, and ABH1, regulate the important steps of the ABA signal transduction pathways (Finkelstein et al., 2002; Leonhardt et al., 2004). Several positive regulators in ABA signaling, such as SRK2E/OST1 and RCN1, have been identified (Kwak et al., 2002; Mustilli et al., 2002; Yoshida et al., 2002). These negative regulators are known to govern the ABA signaling cascades in normal growth conditions. On the other hand, when ABA is synthesized under stress conditions, positive regulators are activated and transmit the signal to downstream pathways (Finkelstein et al., 2002; Leonhardt et al., 2004). These findings and our results suggest that RPK1 functions at early steps of the ABA signal transduction pathway, so that it can regulate many ABA-responsive genes that are controlled through various ABA-dependent signal transduction pathways. Interestingly, the downregulated genes in the microarray analysis of the RPK1 mutants and antisense transgenic plants also included several genes that were not inducible by ABA application, such as reactive oxygen species–related genes (Figure 5, Table 1). Crosstalk between ABA signaling and other signaling seems to exist and affect gene expression patterns in the RPK1 signal transduction pathway. The genes downregulated in the antisense-RPK1 suspension cells were different from those downregulated in the antisense-RPK1 transgenic plants; this difference may be due to cell specificity in plants and suspension cells.
Previous studies have shown that signaling events occur at the plasma membrane in extracellular ABA perception (Yamazaki et al., 2003). We showed the localization of RPK1-GFP at the plasma membrane in Arabidopsis cells (Figure 1). From the localization of the RPK1 protein and global effects in ABA responses in loss-of-function mutants of RPK1, we can speculate that RPK1 binds ABA directly or indirectly and functions as a positive factor in ABA signaling, which governs many ABA-related cellular and molecular events. Another possibility is that RPK1 functions in the perception of unknown secondary messengers or peptide hormones involved in early ABA signaling.
RPK1 gene expression was specifically upregulated by ABA treatment, and phenotypes of RPK1 loss-of-function mutants were observed in the presence of exogenous ABA, suggesting that RPK1 functions effectively at specific growth stages at which ABA operates, such as during seed dormancy and under water stress. Recently, ABA hypersensitivity of a T-DNA insertion mutant of a putative G protein–coupled receptor gene, GCR1, was reported (Chen et al., 2004; Pandey and Assmann, 2004). GCR1 was considered as a possible negative regulator of GPA1-mediated ABA responses (Wang et al., 2001b) via GA and BR signal transduction (Chen et al., 2004). The diverse responsiveness of heterotrimeric G proteins (Jones, 2002) and GCR1 to several phytohormones in Arabidopsis suggested that the GCR1-related ABA signaling system may not directly associate with the RPK1-mediated ABA signaling cascades under ABA-accumulated conditions in Arabidopsis. Possible interaction between GCR1 and sphingosine-1-phosphate or ABA receptors has been suggested (Pandey and Assmann, 2004). These results suggest that several kinds of ABA receptor complex might exist and be activated in various cell- or stage-specific responses to ABA in Arabidopsis cells.
Plant RLK proteins contain a ligand binding extracellular domain, a single membrane-spanning domain, and a cytoplasmic Ser/Thr kinase domain, and many classes of the genes are encoded in the plant genome (Shiu and Bleecker, 2001). More than 600 RLKs in the Arabidopsis genome contain various types of intracellular domains. The RLK extracellular domains are thought to function in their specific ligand bindings. The functional roles of several RLKs have been shown; they can perceive extracellular ligands, including hormones, and regulate downstream cascades and cellular events (Morris and Walker, 2003; Tichtinsky et al., 2003). The RPK1 protein contains two LRR amino acid sequences in its extracellular domain (Hong et al., 1997). The RPK1 gene family in Arabidopsis genome contains two related genes (RPK1 and AT3g02130) that show the high sequence similarity in the kinase domain but not in the LRR domain (Shiu and Bleecker, 2001). To characterize the function of the RPK1 LRR domain, we generated transgenic plants overexpressing a truncated RPK1 protein containing the LRR domain and the membrane-spanning domain (RPK1-LRR, Figure 6). The transgenic plants with RPK1-LRR showed ABA insensitivity in seed germination and growth (Figure 6). The results suggest that the RPK1-LRR domain plays an important role in the perception of an ABA signal.
To further analyze the function of RPK1-LRR, we generated transgenic plants overexpressing the chimeric protein RL-BK, a fusion of RPK1-LRR and BRI1-KD (Figure 7). The function of the BRI1-LRR domain has been confirmed in the characterization of a chimeric protein formed from BRI1-LRR and the kinase domain of a rice (Oryza sativa) disease resistance LRR-RLK, Xa21 (He et al., 2000). The chimeric receptor initiated defense responses in rice cells upon treatment with BR, showing that BRI1-LRR is essential for BR perception (He et al., 2000). Further analysis has detected BR binding activity of the BRI1-LRR domain (Wang et al., 2001a). From these results, we first expected that RL-BK would initiate the BR signal transduction pathway upon treatment with ABA. However, the seed germination of these plants showed increased sensitivity to ABA (Figure 7D). Overexpression of RL-BK also caused a dominant-negative effect on BR signal transduction and strong dwarf phenotypes. Many BR-responsive genes were downregulated in the transgenic plants (Table 2). It is known that the repression of BR signal transduction causes ABA hypersensitivity (Steber and McCourt, 2001). However, the dominant-negative effect was controlled in an ABA-dependent manner in the transgenic plants overexpressing RL-BK (Figure 7). Furthermore, the microarray analysis showed that many ABA-responsive genes included in the RPK1 signal transduction pathway were upregulated in the transgenic plants overexpressing RL-BK (Table 3). These results suggest that the specific RPK1-mediated ABA signal transduction pathway was activated in the RL-BK transgenic plants. RL-BK overexpression in the transgenic plants showed modulated ABA and BR responses through the ABA signal perception of RPK1-LRR.
It has been shown that a BR receptor LRR-RLK, BRI1, requires heterodimerization with another LRR-RLK, BAK1, for its receptor function (Li et al., 2002; Nam and Li, 2002; Li, 2003). Another LRR-RLK, CLV1, which regulates meristem development, can form a heterodimer with an LRR-containing protein, CLV2 (Jeong et al., 1999). This suggests that plant RLKs form dimers or tetramers to become active and transduce the ligand binding signal to the downstream cascades. Isolation and characterization of the RPK1-specific partner in its ABA signaling complex will elucidate the mechanisms of ABA signal perception of RPK1. We are now performing yeast two-hybrid screening to identify RPK1-interacting proteins.
In conclusion, we showed that an RLK, RPK1, functions in ABA signal transduction pathways such as seed germination, stomatal closure, root growth, cell proliferation, and gene expression. RPK1 has a transmembrane domain and is localized on the plasma membrane. The LRR domain of RPK1 may function in early ABA signal perception and constitute an ABA receptor complex. We are now analyzing the ABA binding ability of the RPK1 protein to provide us with more information on ABA perception.
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2500 lux at 22°C. Transgenic plants and control plants were grown on GM agar plates containing kanamycin (20 mg/L). Arabidopsis (Col) T87 cells were maintained in JPL medium (Takahashi et al., 2001) by gentle agitation under continuous illumination of 2500 lux at 22°C. The rpk1 T-DNA knockout of Arabidopsis was isolated by screening the University of Wisconsin Biotechnology Center Arabidopsis Knockout Facility collection of 72,960 BASTA-resistant T-DNA mutant lines (ecotype Ws). A gene-specific primer, 5'-GATCTGCACAAGTACAACTGTGATTCCTT-3', located 1029 bp upstream of the ATG codon of RPK1, and a T-DNA specific primer, 5'-CATTTTATAATAACGCTGCGGACATCTAC-3', were used for screening. The position of the T-DNA insert was determined by sequencing of PCR products carrying the T-DNA and genome junctions. After establishing the presence of homozygotes, we confirmed by a backcross that rpk1-1 was recessive and had a single insertion of T-DNA.
RNA and Protein Gel Blot Analyses
Total RNA was prepared by the method described previously (Osakabe et al., 2002) from Arabidopsis plants grown on GM agar plates for 2 weeks or T87 cells grown in JPL medium for 4 d and treated with or without 10 or 100 µM ABA. Total RNAs were also extracted from wild-type plants treated with distilled water or the plant hormones ABA (Sigma-Aldrich, St. Louis, MO), ethephon (2-chloroethyl-phosphonic acid; Sigma-Aldrich), MeJA (Wako, Osaka, Japan), 2,4-D (Sigma-Aldrich), BA (Sigma-Aldrich), GA3 (Sigma-Aldrich), and SA (Sigma-Aldrich). Thirty micrograms of total RNA was fractionated in a 1% agarose gel containing formaldehyde and blotted onto a nylon membrane. The membrane was then hybridized with a 32P-labeled RPK1 cDNA fragment or full-length cDNA (Seki et al., 2002b) as a probe. Hybridization was performed in 50% formamide, 5x SSC (1x SSC is 0.15 M NaCl and 0.015 M sodium citrate), 25 mM sodium phosphate buffer, pH 6.5, 10x Denhardt's solution (1x Denhardt's solution is 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% BSA), and 250 µg/mL denatured salmon sperm DNA at 42°C for overnight. The membranes were then washed three times with 0.1x SSC and 0.1% SDS at 60°C for 15 min and subjected to autoradiography.
Crude proteins were extracted from wild-type or knockout plants grown on GM agar plates for 3 weeks or from T87 transgenic and wild-type cells grown for 4 d in JPL medium treated with/without 100 µM ABA, then separated by SDS-PAGE and blotted onto a phenylmethylsulfonyl fluoride membrane. RPK1 protein was detected with anti-RPK1 peptide antibody with the ECL Plus protein gel blotting detection aystem (Amersham Biosciences, Piscataway, NJ).
ABA Sensitivity
ABA-sensitive assays were performed as follows. Seeds of antisense-RPK1, rpk1-1, RPK1-LRR ox, and 35S:RPK1rpk1-1 that carried the 35S:RPK1 full-length cDNA in rpk1-1 and seeds of control plants were sterilized and plated on GM medium containing 1% sucrose and 0 or 0.5 µM ABA. Seeds were grown under continuous light at 22°C. To test the ABA sensitivity of root growth, seeds of the mutants and wild-type plants were grown on GM medium for 6 d. After germination, the seedlings were transferred to fresh GM medium containing various concentrations of ABA. After 10 d of culture, the root lengths were measured. For stomatal movement assay, detached rosette leaves of 4-week-old rpk1 knockouts and control plants grown on soil were incubated for 2 h in 0, 2, and 5 µM ABA solution containing 20 mM KCl, 1 mM CaCl2, and 5 mM Mes-KOH, pH 6.15 (Pei et al., 1998). Guard cells were photographed under a color laser three-dimensional profile microscope (Keyence, Osaka, Japan).
Construction of Transgenic Plants
The sense or antisense full-length cDNA fragment of RPK1 was cloned into the pBE2113 vector (Mitsuhara et al., 1996), which contains the modified 35S promoter of Cauliflower mosaic virus and the 
sequence of the Tobacco mosaic virus. For construction of the RPK1-GFP fusion, we used the subcloned RPK1 cDNA as a template and 5'-TGTGAAGAAAAAGATCTAGAATGAAACTTC-3' as a vector primer to amplify the 1617-bp fragment (from ATG to an internal XbaI site). The fragment was then digested with XbaI and cloned into the XbaI site of the pBI121-sGFP (S65T) vector. To amplify the 815-bp promoter region of the RPK1 gene, we used 5'-GAGGAATACACGTCAAGCTTAAATAAAAAT-3' and 5'-GGATCCCAGAAGTTTCATTTTTCTTCTTTT-3', and the amplified fragment was cloned into the pBI101 vector. The constructs were then introduced into Agrobacterium tumefaciens C58. Six-week-old wild-type Arabidopsis (Col) or rpk1-1 plants were transformed by the vacuum infiltration method. Fifteen antibiotic-resistant transgenic lines carrying each construct were further analyzed. Arabidopsis T87 suspension cells cultured for 3 d were coincubated with Agrobacterium for 2 d to transform the constructions and then selected out on a callus induction medium plate containing kanamycin. The selected calluses were then incubated in JPL medium.
Reporter Gene Assays
The tissue-specific activity of GUS was detected by the overnight incubation of 2-week-old transgenic plants carrying the RPK1 promoter-GUS construct, in a GUS staining buffer (1 mM 5-bromo-4-chloro-3-indolyl-ß-D-glucuronic acid, 0.1 M sodium phosphate buffer, pH 7, and 8 mM ß-mercaptoethanol) at 37°C. Staining was stopped by transferring samples into 70% ethanol, and the chlorophyll was then destained. For GFP analysis, roots from 2-week-old transgenic plants containing 35S:RPK1-GFP or 35S:GFP were examined under a confocal microscope.
Agilent Oligo Microarray Analysis
Five-day-old seedlings of the rpk1-2 and wild-type Ws plants were treated with 10 µM ABA for 5 h. Total RNAs were prepared from the ABA-treated rpk1-2, wild-type Ws plants, the 35S:RL-BK transgenic plants, and the control plants carrying the vector grown on GM agar plates for 3 weeks with Trizol reagent (Gibco BRL, Cleveland, OH) and used for the preparation of Cy5-labeled and Cy3-labeled cDNA probes. Samples were subjected to microarray experiments using the Agilent Arabidopsis 2 Oligo Microarray (Agilent Technologies). All microarray experiments, including the data analysis, were performed according to the supplier's manual (http://www.chem.agilent.com/scripts/generic.asp?lpage=11617andindcol=Nandprodcol=Y). The reproducibility of microarray analysis was assessed by a dye swap in each experiment. Genes showing a signal value <600 in both Cy3 and Cy5 channels of the control plants were not considered for the analysis. Feature extraction and image analysis software (version A.6.1.1; Agilent Technologies) was used to locate and delineate every spot in the array and to integrate each spot's intensity, filtering, and normalization using the Lowess method. Gene clustering analysis was performed with Genespring 6.1 (Silicon Genetics, San Carlos, CA).
RIKEN Arabidopsis Full-Length cDNA Microarray Analysis
Trizol reagent was used to extract total RNA for microarray analysis from VC or antisense-RPK1 transgenic plants or T87 cells treated with 10 µM ABA for 5 h. mRNA was prepared with an MACS mRNA isolation kit (Miltenyi Biotec, Bergisch, Gladbach, Germany). mRNAs from the control and antisense plants (antisense T87 cells) were used for preparation of Cy5-labeled and Cy3-labeled cDNA probes, respectively. These cDNA probes were mixed and hybridized with 7K RIKEN Arabidopsis full-length cDNA microarray containing 7000 Arabidopsis cDNAs. The information on the 7K RIKEN Arabidopsis full-length cDNA microarray is available at http://pfgweb.gsc.riken.go.jp/projects/microarray.html. A 
control template DNA fragment (TX803; Takara, Tokyo, Japan) was used as an external control to equalize hybridization signals generated from different samples. To assess the reproducibility of microarray analysis, each experiment was repeated three times. Genes showing a signal value <1000 (which was typically twice the mean background value) in control were not considered for the analyses. The image analysis and signal quantification were performed with QuantArray version 2.0 (GSI Lumonics, Oxnard, CA). Gene clustering analysis was performed with Genespring (Silicon Genetics).
BRI1 Gene Expression Analysis
The BRI1 gene expression levels in the RL-BK transgenic plants were detected by quantitative RT-PCR. Total RNAs were isolated from the 14-d-old dwarf and normal plants of the transgenic lines (L2 and L3) and control plants with Trizol reagent (Gibco BRL). Reverse transcription of all the RNA samples was performed with SuperScript III RNase H reverse transcriptase (Invitrogen, Carlsbad, CA). SYBR Premix Ex Taq (Takara) was used for real-time quantitative PCR. We used LightCycler3 (Roche Diagnostics, Indianapolis, IN) and repeated experiments three times. For detection of Bri1, PCR primers 5'-CATTCTTGATCTTTCAAGCAATAAGCTCGATGGG-3' and 5'-ATCCCATCGCCACACTACCAGCAAGGGACG-3' were used. Melting curves confirmed that only one product was amplified. Specific cDNA was quantified with a standard curve based on known amounts of amplified BRI1 fragment. The 18S rRNA was also amplified and used for calibration.
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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.104.027474.
Received August 31, 2004; accepted January 16, 2005.
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