First published online April 3, 2003; 10.1105/tpc.010413
The Plant Cell, Vol. 15, 1095-1110,
May 2003, Copyright © 2003,
American Society of Plant Biologists
Dominant-Negative Receptor Uncovers Redundancy in the Arabidopsis ERECTA Leucine-Rich Repeat Receptor–Like Kinase Signaling Pathway That Regulates Organ Shape
Elena D. Shpaka,
Michael B. Lakemana and
Keiko U. Torii1,a,b
a Department of Biology, University of Washington, Seattle, Washington 98195-1800
b CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 322-0012, Japan
1 To whom correspondence should be addressed. E-mail ktorii{at}u.washington.edu; fax 1-206-685-1728
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Abstract
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Arabidopsis ERECTA, a Leu-rich repeat receptor-like Ser/Thr kinase (LRR-RLK), regulates organ shape and inflorescence architecture. Here, we show that a truncated ERECTA protein that lacks the cytoplasmic kinase domain ( Kinase) confers dominant-negative effects when expressed under the control of the native ERECTA promoter and terminator. Transgenic plants expressing Kinase displayed phenotypes, including compact inflorescence and short, blunt siliques, that are characteristic of loss-of-function erecta mutant plants. The Kinase fragment migrated as a stable  400-kD protein complex in the complete absence of the endogenous ERECTA protein and significantly exaggerated the growth defects of the null erecta plants. A functional LRR domain of Kinase was required for dominant-negative effects. Accumulation of Kinase did not interfere with another LRR-RLK signaling pathway (CLAVATA1), which operates in the same cells as ERECTA but has a distinct biological function. Both the erecta mutation and Kinase expression conferred a lesser number of large, disorganized, and expanded cortex cells, which are associated with an increased level of somatic endoploidy. These findings suggest that functionally redundant RLK signaling pathways, including ERECTA, are required to fine-tune the proliferation and growth of cells in the same tissue type during Arabidopsis organogenesis.
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INTRODUCTION
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The bodies of higher plants are built by a reiterative formation of the shoot system, which consists of a node bearing a lateral organ (e.g., a leaf) and an internode (e.g., a stem). Both lateral organs and internodes are generated from distinct domains of the shoot apical meristem, in which continual cell proliferation and differentiation take place. The basic pattern and identity of the shoots are determined at the shoot apical meristem, and their final size and shape, which contribute to the diversity of the plant form, are elaborated by localized cell division and cell expansion during plant organ morphogenesis.
Although mechanisms for plant cell division and expansion have been studied extensively, little is known about how these two cellular processes are integrated in the context of whole plant growth and development. Transgenic studies of cell cycle regulators revealed intimate crosstalk between these two processes. For instance, transgenic tobacco plants overexpressing a dominant-negative form of Cdc2 produced nearly normal organs, both in overall size and patterning, despite the fact that the transgene severely compromised cell division (Trotochaud et al., 1999 ). Although overexpression of the cyclin kinase inhibitor ICK1 in Arabidopsis plants resulted in small organs, the cells that made up such small organs were much larger than control cells (Wang et al., 2000). These findings imply that plants may somehow monitor and balance the activity of cell division and cell expansion to retain a stable organ size. Therefore, revealing the mechanisms that coordinate these two processes at the supracellular level is critical for a better understanding of plant growth and development.
The molecular basis of the cell-to-cell signaling that coordinates cell division and expansion during plant organogenesis is not clear. One candidate gene is Arabidopsis ERECTA, which regulates organ shape and inflorescence architecture. Loss-of-function erecta mutations confer a compact inflorescence with short internodes and clustered flower buds, short pedicels, round flowers, and short, blunt siliques (Bowman, 1993; Torii et al., 1996). Despite these defects, the erecta mutation does not affect organ identity, polarity, or tissue organization. As such, Landsberg erecta has been used widely as a "wild type" because of its preferable, compact plant size. Cellular defects caused by erecta are not documented extensively; however, both cell size and number are altered in erecta inflorescence stems (Komeda et al., 1998). Consistent with its role in organogenesis, ERECTA is expressed at high levels in the entire shoot apical meristem and developing organs (Yokoyama et al., 1998). ERECTA encodes a Leu-rich repeat receptor-like kinase (LRR-RLK) with functional Ser/Thr kinase activity (Torii et al., 1996; Lease et al., 2001a). The LRR-RLKs constitute the largest subfamily of plant RLKs and possess a structural organization similar to that of animal receptor kinases (Torii, 2000; Shiu and Bleecker, 2001). Several LRR-RLKs function as important developmental regulators, including Arabidopsis CLAVATA1 (CLV1), which balances cell proliferation and differentiation in the meristem, RLK5/HAESA, which promotes flower abscission, and the brassinosteroid (BR) receptor BRI1 (Clark et al., 1997; Li and Chory, 1997; Jinn et al., 2000). The mutant phenotypes, expression patterns, and molecular identity of ERECTA as an LRR-RLK support the notion that ERECTA mediates yet to be identified cell-to-cell signaling pathways that coordinate shoot organ growth.
To understand how ERECTA regulates organ growth, it is essential to elucidate its modes of action as a receptor kinase and to identify other components of the ERECTA-mediated signal transduction pathway. A large-scale genetic screen searching for the erecta phenotype led to the isolation of 16 additional erecta alleles but failed to identify novel loci whose mutations confer phenotypes identical to that of erecta (Lease et al., 2001a, 2001b). This finding implies that other components of the ERECTA signaling pathway are redundant. Therefore, their mutations may not have an erecta-like phenotype; instead, they may display no phenotype at all, a subset of the erecta phenotypes, more severe phenotypes, or even be lethal. From the same genetic screen, erecta-like4 (elk4) was identified as a silique-specific genetic interactor of erecta, suggesting that ELK4 may be a tissue-specific component of the ERECTA signaling pathway (Lease et al., 2001b). ELK4 encodes AGB1, a putative  -subunit of the heterotrimeric G-proteins, which are known to transduce signals via seven transmembrane G-protein–coupled receptors in animals (Lease et al., 2001b). It remains to be determined how heterotrimeric G-proteins interact with the ERECTA LRR-RLK signaling pathway.
We sought to dissect the ERECTA signaling pathway using a transgenic approach. Expression of truncated receptor kinases has been used widely as a powerful tool to reveal in vivo function and signal transduction of animal receptor kinases. For both animal receptor Tyr kinases (RTK) and transforming growth factor receptor Ser/Thr kinases, the general consensus is that truncated receptors that lack the cytoplasmic kinase domain act as dominant-negative receptors by blocking the normal activity of the endogenous counterparts (Amaya et al., 1991; Ueno et al., 1991; Hemmati-Brivanlou and Melton, 1992; Freeman, 1996). Such an approach has not been pursued actively in plant RLK studies because of frequent cosuppression events (Conner et al., 1997; Schumacher and Chory, 2000). An additional confusion in understanding the modes of action of the plant RLK is that the kinase domain of LRR-RLK appears dispensable, because truncated mutations that remove the entire kinase domain of two LRR-RLKs, CLV1 and rice Xa21, retain partial activity (Clark et al., 1997; Wang et al., 1998; Torii, 2000).
Here, we report that truncated ERECTA protein that lacks the cytoplasmic kinase domain (Kinase) interferes with endogenous ERECTA function. Therefore, unlike CLV1 and Xa21, Kinase of ERECTA acts as a dominant-negative receptor. Importantly, the Kinase protein enhances the phenotype of the null erecta plants. Kinase migrates as an 400-kD protein complex in the absence of the endogenous ERECTA protein, suggesting that Kinase associates with other RLKs and/or ligands, which are shared by other RLKs, and blocks their functions. Based on cell biological analysis of the erecta mutants and Kinase transgenic plants, we propose that multiple overlapping and interrelated RLK signaling pathways, including ERECTA, are required for coordinated cell proliferation and cell growth within the same tissue types during Arabidopsis organogenesis.
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RESULTS
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Transgenic Plants That Express Kinase Display the erecta Mutant Phenotype
We introduced into Arabidopsis wild-type ERECTA plants (ecotype Columbia) a truncated ERECTA that retains the extracellular LRR and transmembrane domains but lacks the cytoplasmic kinase domain (Kinase). To ensure the proper temporal/spatial expression patterns of the truncated ERECTA, the 1.7-kb 5' and 1.9-kb 3' regions of the Columbia ERECTA locus, which correspond to the ERECTA promoter and terminator, respectively (Yokoyama et al., 1998), as well as a genomic fragment of Kinase, which contains all 23 introns (Torii et al., 1996), were used to express Kinase. The Kinase fragment contains a short cytoplasmic tail of 12 amino acids, which is juxtaposed to the putative transmembrane domain. Fifty-one of 54 independent T1 plants showed a phenotype resembling that of loss-of-function erecta mutant plants (Figure 1). Analysis of the selfed T2 progeny revealed that the phenotype was dominant and linked tightly to the transgene (data not shown). Among the lines that contained a single T-DNA insertion, two lines (L1 and L2) with strong phenotype and one line (L3) with mild phenotype were chosen for a further characterization.
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Figure 1. ERECTA::Kinase Confers Dominant-Negative Effects in Arabidopsis Morphology.
(A) Seven-week-old plants of the wild type (WT), Kinase, and erecta-105. Bar = 4 cm.
(B) Inflorescence apices from the wild type, Kinase, and erecta-103. Bars = 3 mm.
(C) Scanning electron micrographs of silique tips from the wild type, Kinase, and erecta-105. Bars = 100 µm.
(D) Siliques and attached pedicels of the wild type, Kinase, and erecta-105. Bar = 5 mm.
(E) Morphometric analysis of fully grown 7-week-old plants of the wild type, erecta-105, and three independent transgenic lines (L1, L2, and L3) of ERECTA::Kinase. Twenty-five plants were analyzed for plant (inflorescence) height. Lengths of 50 mature pedicels and siliques on the main inflorescence stem (10 measurements per stem) were analyzed. Bars represent average values ± SD.
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Transgenic ERECTA::Kinase plants had short stature and developed a compact inflorescence, short pedicels, and short, blunt siliques, all of which are reminiscent of loss-of-function erecta mutant plants (Figures 1A to 1D). The ERECTA::Kinase inflorescence tip displayed a characteristic clustering, which was indistinguishable from that of the intermediate allele erecta-103 (Figure 1B) (Torii et al., 1996). Detailed morphometric analysis revealed that plant height and pedicel length of ERECTA::Kinase plants were intermediate between those of the wild type and the null allele erecta-105, with L3 being tallest (Figure 1E). Silique lengths of all three lines were as short as that of erecta-105 (Figure 1E). The morphology of the silique tips was analyzed in detail (Figure 1C). The tip of the wild-type silique had an elongated style that protruded from narrow valves. By contrast, the tips of the erecta and ERECTA::Kinase siliques (L2) had short and broad styles (Figure 1D). These results suggest that the organ elongation defects conferred by ERECTA::Kinase highly resemble the disruption of normal ERECTA function. The transgenic plants also displayed reduced fertility as a result of defective elongation of the stamen filaments (data not shown).
Accumulation of the Kinase Fragment Confers Dominant-Negative Interference
The erecta phenotype conferred by the introduction of ERECTA::Kinase could be attributable to dominant-negative interference of the ERECTA pathway by the truncated ERECTA receptor. Alternatively, it could be the result of the cosuppression of endogenous ERECTA gene expression. To distinguish between these two possibilities, we next examined the level of the endogenous ERECTA transcripts in three transgenic lines. Reverse transcriptase–mediated (RT) PCR analysis using primers that anneal to the kinase domain revealed that levels of the endogenous ERECTA transcripts were not altered significantly by the transgene, excluding the possibility of cosuppression (Figure 2A).
From immunoblots probed with antibody raised against the ERECTA LRR domain (anti-ERLRR), endogenous ERECTA was detected as a band of 145 kD in both wild-type and transgenic plants (Figure 2B). In the erecta-105 null allele background, we detected a very faint band at a similar position (Figure 2B), likely representing LRR-RLKs closely related to ERECTA. The higher molecular mass of ERECTA compared with its calculated molecular mass (105 kD) suggests a possible glycosylation, because the extracellular domain of ERECTA possesses 12 potential N-glycosylation sites (Torii et al., 1996). Several other plant LRR receptors, including tomato Cf-4/Cf-9 and the carrot phytosulfokine receptor, have been shown to be glycosylated (Piedras et al., 2000; Matsubayashi et al., 2002; Rivas et al., 2002a, 2002b). The Kinase protein migrates at 95 kD (the predicted polypeptide is 64 kD) and therefore may be glycosylated as well (Figure 2B).
Interestingly, we found that the Kinase protein was accumulated at much higher levels than the full-length, endogenous ERECTA protein (Figure 2B). A quantitative analysis of the immunoblot signals estimated that the amount of Kinase was 100 times greater in L1 and L2 and 30 times greater in L3 than the endogenous ERECTA (Figure 2B). RT-PCR analysis with primers that amplify both endogenous and truncated ERECTA revealed that the amounts of Kinase transcripts were at levels comparable to those of the endogenous ERECTA transcripts (Figure 2A). Therefore, the increased amount of Kinase was associated with post-transcriptional regulation, most likely as a result of the increased stability of the truncated protein. From these results, we conclude that the observed erecta phenotype of ERECTA::Kinase transgenic plants most likely is conferred by dominant-negative interference of highly stable Kinase protein with the endogenous ERECTA pathway.
Both RT-PCR with primers specific to the Kinase transgene and immunoblot analysis demonstrated that ERECTA::Kinase was expressed at a level three times greater in the lines with severe phenotype (L1 and L2) than in the line with mild phenotype (L3) (Figures 2A and 2B). Thus, the phenotypic severity correlates with the amount of Kinase gene products in a dosage-dependent manner.
A Kinase Fragment Further Enhances the Growth Defects in the Null Allele of erecta
The dominant-negative effects of Kinase were quite surprising, given that similar deletion mutants in CLV1 and Xa21 (e.g., clv1-6 and Xa21-D) have been shown to retain partial function (Clark et al., 1997; Wang et al., 1998; Torii, 2000). Therefore, we sought to determine the underlying mechanism of the dominant-negative interference. If the ERECTA signaling pathway is strictly homodimeric and linear, we predict that the expression of Kinase will not enhance the erecta null phenotype. By contrast, Kinase may make the erecta null phenotype even more severe if the ERECTA signaling pathway is redundant. To address these hypotheses, we introduced ERECTA::Kinase into erecta-105 plants, which do not produce any ERECTA transcripts (Torii et al., 1996; Lease et al., 2001b). Expression of Kinase in erecta-105 conferred severe growth defects (Figures 3 and 4). The transgenic plants were dwarf, with extremely short internodes, pedicels, and siliques as well as smaller, round flowers (Figures 3A to 3C). The development of flower organs seemed less coordinated, and pistils protruded above buds (Figure 3B).
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Figure 3. Expression of Kinase Enhances the Growth Defects in the Null erecta Allele.
(A) Seven-week-old plants of erecta-105, Kinase/erecta-105, and Kinase–c-Myc/erecta-105. Bar = 4 cm.
(B) Inflorescence apices from erecta-105, Kinase/erecta-105, and Kinase–c-Myc/erecta-105. Bars = 3 mm.
(C) Siliques and attached pedicels of erecta-105, Kinase/erecta-105, and Kinase–c-Myc/erecta-105. Bar = 5 mm.
(D) Morphometric analysis of fully grown 7-week-old plants of erecta-105, three independent transgenic lines of ERECTA::Kinase/erecta-105 (L1, L2, and L3), and one line of ERECTA::Kinase–c-Myc/erecta-105. Plant (inflorescence) height, pedicel length, and silique length were measured as described for Figure 1E. The length of the siliques was measured in only one line of ERECTA::Kinase/erecta-105 (L3), because the other two lines had reduced fertility as a result of short filaments. Bars represent average values ± SD.
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ERECTA::Kinase–c-Myc, which contains a triple c-Myc sequence (EQKLISEEDL) at the end, retained the ability to exaggerate the erecta null phenotype, albeit slightly less effectively (Figures 3A to 3D). This finding could be attributable to steric hindrance by the triple c-Myc peptides or, alternatively, to reduced accumulation of the gene products (Figure 4B). Immunoblot analysis revealed that Kinase–c-Myc was detected by both anti-ERLRR and anti-c-Myc antibodies as a band of 105 kD, slightly larger than the nontagged Kinase (Figure 4B). The anti-c-Myc antibody was highly specific and essentially gave no background signal (Figure 4B).
Although both Kinase and Kinase–c-Myc confer phenotypes much more severe than that of erecta-105 or any of the available 24 erecta alleles (Lease et al., 2001a), the phenotypic characteristics, such as compact inflorescence and short, blunt siliques, were consistent with the erecta defects (Figure 3). Together, these data support our hypothesis that nonfunctional Kinase is capable of interfering with and shutting down the ERECTA and related RLK pathways that regulate organ elongation in a partially redundant manner (see Discussion).
The Highly Accumulated ERECTA Kinase Fragment Does Not Interfere with the CLV1 LRR-RLK Signaling Pathway
Use of the endogenous ERECTA promoter and terminator should minimize the ectopic effects of Kinase. However, it is possible that a highly stable Kinase fragment associates in a nonspecific manner with RLKs that are expressed in the same tissue/cell types as ERECTA but that do not normally interact with the ERECTA signaling pathway. We sought to determine whether the dominant-negative Kinase inhibits a well-studied RLK signaling pathway that has overlapping expression patterns with ERECTA.
For this purpose, we investigated whether ERECTA::Kinase inhibits the CLV1 signaling pathway. CLV1 is expressed at the subepidermal layers in the center of the shoot and flower meristems, whereas ERECTA is expressed broadly within these meristems (Clark et al., 1997; Yokoyama et al., 1998); therefore, Kinase likely accumulates in the cells in which CLV1 normally functions. The hallmark of the clv phenotype is an increased number of floral organs as a result of enlarged floral meristems (Leyser and Furner, 1992; Clark et al., 1993). Although the wild-type flower has two carpels, average carpel numbers of the severe allele clv1-4 and the weak allele clv1-6 are 5.12 ± 0.04 and 3.91 ± 0.04, respectively (Yu et al., 2000). As shown in Figure 5A, carpel numbers were not affected by the expression of ERECTA::Kinase. Wild type, erecta-105, Kinase in wild-type, and Kinase in erecta-105 all produced siliques with two carpels (2.00 ± 0.00, n = 40 for each genotype).
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Figure 5. Expression of Kinase Does Not Interfere with the CLV Signaling Pathway, and ERECTA Does Not Associate with KAPP, a Negative Regulator of Multiple RLKs, Including CLV1.
(A) Number of carpels in wild-type (WT), erecta-105, ERECTA::Kinase/wild type (L1), ERECTA::Kinase/erecta-105 (L3), and ERECTA::Kinase–c-Myc/erecta-105 flowers. Bars represent mean values (n = 40) for each genotype.
(B) Semiquantitative RT-PCR analysis of WUS transcripts from buds and flower clusters of erecta-105 and three independent lines of ERECTA::Kinase/erecta-105 plants. WUS expression levels are unaltered by the Kinase fragment.
(C) KAPP KID does not associate with an active kinase catalytic domain of ERECTA. Top, Coomassie blue–stained SDS-PAGE gel of the affinity-purified, recombinant ERECTA and RLK5/HAESA kinase domains, both active (WT) and inactive (for ERECTA, K676E; for RLK5, K711E) forms, fused to the maltose binding protein. One microgram of the purified proteins was loaded on the gel. Molecular mass markers are indicated at left. Bottom, Autoradiogram of the purified proteins dot-blotted onto a polyvinylidene difluoride membrane and probed with the 32P-labeled GST-KID fusion protein.
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Molecular-genetic studies have shown that the CLV signaling pathway restricts the expression domain of WUSCHEL (WUS), which specifies stem cell fate (Brand et al., 2000; Schoof et al., 2000). Unlike clv mutations, which confer ectopic upregulation of WUS expression (Brand et al., 2000; Schoof et al., 2000), our semiquantitative RT-PCR analysis revealed that ERECTA::Kinase had no effect on WUS expression levels (Figure 5B). From these phenotypic and molecular analyses, we conclude that highly accumulated Kinase does not interfere with the CLV signaling pathway.
These findings suggest that the components of ERECTA and CLV signaling pathways are quite distinct, even though the structural features of these two LRR-RLKs are similar. Therefore, we investigated whether ERECTA associates with a known component of the CLV pathway, Kinase-Associated Protein Phosphatase (KAPP). KAPP associates with the kinase domains of several RLKs, including RLK5/HAESA and CLV1, via its kinase interaction domain (KID) (Stone et al., 1994, 1998; Williams et al., 1997). We expressed both wild-type and kinase-inactive forms of ERECTA fused to the maltose binding protein. The kinase-inactive version (K676E) has a substitution of an invariant Lys in subdomain II to Glu. The interaction of ERECTA with the KAPP KID was tested by dot-blot analysis, because it gave stronger signals than electroblotted samples. As shown in Figure 5C, positive signal was detected only in the kinase-active form of RLK5/HAESA, which was used as a positive control. Because ERECTA possesses Ser/Thr protein kinase activity (Lease et al., 2001a), we conclude that ERECTA does not associate with KAPP in vitro. These results indicate that the expression of the Kinase fragment of ERECTA does not affect the CLV1 pathway, which most likely operates in a distinct manner; they further imply that the dominant-negative effects of Kinase involve specific mechanisms.
A Functional LRR Domain of ERECTA Is Required for the Dominant-Negative Effects
To gain more insight into the mechanisms of Kinase action, we introduced into the Kinase fragment a point mutation corresponding to erecta-103, which replaces the Met within the 10th LRR with Ile (M282I) (Torii et al., 1996) (Figure 6A). Introduction of the mutation did not result in reduced stability of the transcripts or proteins; instead, the amount of the KinaseM282I protein appeared to have increased slightly (Figure 6B). Nevertheless, the M282I mutation severely compromised the dominant-negative effects of Kinase, because transgenic erecta-105 plants expressing KinaseM282I no longer displayed severe dwarfism, extreme compact inflorescence, or reduced fertility (Figure 6C). These results indicate that a functional LRR domain is required for the dominant-negative interference and imply that structural integrity of the extracellular LRR domain may be crucial for titrating ligand or receptor partners that are shared by RLKs, which possess overlapping function with ERECTA.
Dominant-Negative Kinase Migrates as a Protein Complex in the Absence of Endogenous ERECTA
If Kinase confers dominant-negative interference through direct association with the components (such as ligands and partners) of related RLKs, we would expect Kinase to form a protein complex. To test this hypothesis, we investigated the behavior of Kinase by gel-filtration chromatography. Flowers and bud clusters of transgenic erecta-105 plants expressing either Kinase or Kinase–c-Myc were used as materials to minimize the complications of having both full-length and truncated ERECTA. In the presence of 1% Triton X-100, the Kinase protein migrated as a complex of 400 kD (Figure 7, top gel). No signal of similar size was detected in the control erecta-105 fractions (Figure 7, middle gel). Some Kinase may exist at 100 kD, representing monomers. The presence of nonspecific signals of similar size in the control erecta-105 fractions, however, makes this possibility inconclusive (Figure 7). The immunoblot probed with anti-c-Myc antibodies revealed that Kinase–c-Myc migrated exclusively as a complex of a similar size with a slightly broader range of elution, which may be the result of less efficient interactions of Kinase–c-Myc with other components caused by steric hindrance (Figure 7, bottom gel). The fact that Kinase migrated as a protein complex in the absence of the endogenous ERECTA is consistent with our hypothesis that the physical interaction of nonfunctional Kinase with the other RLKs enhances the organ elongation defects in erecta-105.
ERECTA Regulates Proper Cell Proliferation and Polarity
To understand how ERECTA controls organ elongation, we analyzed the cellular defects in erecta and in dominant-negative transgenic plants. Mature pedicels were examined, because the degree of allelic severity correlates with reduction in pedicel length (Torii et al., 1996; Lease et al., 2001a). Figure 8 shows representative, longitudinal sections of fully expanded mature pedicels. Although the wild-type pedicels were approximately twice as long as erecta-105 pedicels (Figure 1D), cells in the cortex and endodermis of erecta-105 were notably larger than wild-type cells, indicating that the short pedicel phenotype is attributable to fewer cells (Figures 8A and 8C). Moreover, cortex cells were expanded radially, accounting for the thick pedicel phenotype of erecta (Figure 8C). The epidermal cells of erecta-105 were slightly shorter than the wild-type cells. We detected no significant difference in the pith (data not shown). Our observations indicate that erecta is not a typical dwarf mutant with general cell elongation defects.
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Figure 8. erecta Mutation and Kinase Expression Lead to Aberrant Cell Enlargement, Reduced Cell Numbers in the Cortex, and Increased Endoploidy in Mature Pedicels.
(A) to (D) Longitudinal sections of mature pedicels from wild-type (WT) (A), ERECTA::Kinase (B), erecta-105 (C), and ERECTA::Kinase/erecta-105 (D) plants. Photographs were taken at the same magnification. cor, cortex; en, endodermis; ep, epidermis. Bars = 25 µm.
(E) and (F) Flow cytometric analysis of mature pedicels from wild-type, intermediate allele erecta-103, ERECTA::Kinase, null allele erecta-105, and ERECTA::Kinase/erecta-105 plants. (E) shows representative nuclei counts, and (F) shows the 2C/4C ratio as an average of four independent counts. Error bars indicate standard errors.
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Similar to the erecta mutation, the Kinase protein conferred reduced cell numbers associated with enlarged and irregular cell shape in the cortex and endodermis (Figure 8). However, unlike erecta-105, Kinase cortex cells were not expanded laterally. In the ERECTA::Kinase/erecta-105 pedicels, disorganized cell growth in the cortex was even more evident (Figure 8D). Although some cells in the cortex were large and expanded, others remained small, leaving many "gaps" between the cells. These observations suggest that ERECTA and its overlapping pathways are required for coordinated cell proliferation and proper cell–cell interactions within the cortex cell layers.
Increased Levels of Somatic Endoploidy in Pedicels of erecta and Kinase Plants
Increased cell size in general correlates with increased DNA content or ploidy level (Kondorosi et al., 2000). Because mature pedicels of erecta and Kinase have enlarged cortex cells, we measured their ploidy levels to determine whether the inhibition of ERECTA signaling leads to somatic endoploidy. A majority (62%) of nuclei in the wild-type pedicels remained diploid (2C), whereas some were tetraploid (4C; 31%) and a few were octaploid (8C; 7%) (Figure 8E). Both intermediate allele erecta-103 and Kinase pedicels showed increases in the 4C nuclei (37 and 36%, respectively), making the 2C/4C ratio 1.5, in contrast to 2.0 in the wild-type pedicels (Figures 8E and 8F). The 4C nuclei content was highest in the null allele erecta-105 pedicels (49%; 2C/4C ratio = 0.9), whose cortex cells were the largest (Figure 8). Therefore, the degree of erecta defects and cortex cell size have a positive correlation with increased 4C content. The expression of Kinase in erecta-105 did not confer an additional increase in 4C content (47%; 2C/4C ratio = 0.97) (Figures 8E and 8F). This finding is consistent with our histological observation that Kinase/erecta-105 did not lead to extra cell enlargement but rather disrupted the proper coordination of cortex cell development (Figure 8D). None of the genotypes showed increased amounts of 8C (Figure 8E), indicating that inhibition of the ERECTA pathway does not activate endoreduplication cycles. Because mature pedicels do not express the cell-cycling marker Cyc1At::GUS (K.U. Torii, unpublished data), it is very unlikely that the 4C nuclei represent actively proliferating S-phase cells. Together, our findings suggest that erecta mutations and Kinase expression may inhibit cell division and promote premature differentiation of the 4C cells.
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DISCUSSION
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In this study, we provide evidence that truncated ERECTA lacking the cytoplasmic kinase domain (Kinase) confers dominant-negative effects in Arabidopsis organ growth and internodal elongation. Biochemical and phenotypic analyses of the null erecta allele expressing Kinase revealed the presence of multiple interrelated pathways that act together with ERECTA to coordinate cell proliferation and cell expansion within the same cell types.
Modes of Action of ERECTA and Redundancy in the ERECTA Signaling Pathway Revealed by Kinase Expression
Unlike animal receptor kinases, the kinase domains of some plant LRR-RLKs appear partially dispensable. For instance, two clv1 alleles that truncate the entire kinase domain, clv1-6 and clv1-7, have the weakest phenotypes (Clark et al., 1997). Expression of Xa21D, a naturally occurring variant of the rice disease resistance gene that lacks the entire kinase domain, confers partial resistance to a full spectrum of pathogens (Wang et al., 1998). A model for Xa21D action is that it forms a functional heterodimer with an RLK, which lacks ligand recognition specificity, and that ligand binding to the LRR domain of Xa21D stimulates the phosphorylation of its receptor partner and initiates signaling (Wang et al., 1998). By contrast, expression of the Kinase form of ERECTA resulted in an inhibition of normal ERECTA functions (Figures 1 and 2). Based on the analogy from animal receptor kinase signaling, it is reasonable to hypothesize that Kinase forms a nonfunctional receptor dimer with the endogenous receptor partner of ERECTA and prevents the transphosphorylation that is a prerequisite for the activation of the signaling cascades.
In the absence of endogenous ERECTA protein, Kinase migrates as an 400-kD protein complex, which most likely represents a nonfunctional receptor oligomer (Figure 5), suggesting that ERECTA may function as a heterooligomer. Some plant LRR-RLKs function as heterodimers. For example, CLV1 forms a heterodimer CLV2 LRR transmembrane protein, presumably via disulfide linkage (Jeong et al., 1999; Trotochaud et al., 1999). Recently, the Arabidopsis BAK1 LRR-RLK was identified as a receptor partner of BRI1 in BR signaling (Li et al., 2002; Nam and Li, 2002).
The apparent discrepancy in the recessive nature of erecta mutations and the dominant effects of ERECTA::Kinase can be explained by the high level of accumulation of Kinase protein. Although the mRNA levels of Kinase and endogenous ERECTA are similar, the amount of the Kinase protein is 100-fold greater than that of the full-length ERECTA protein, most likely as a result of increased protein stability (Figures 2 and 4). In animals, ligand-induced degradation of the epidermal growth factor RTK plays an important role in the downregulation of epidermal growth factor signaling (Beguinot et al., 1984; Jones et al., 2002). Similarly, the amount of endogenous ERECTA RLK may be regulated tightly during organogenesis. Perhaps the truncated Kinase is not under such regulation; thus, it stably locks in and sequesters the signaling components.
Although highly accumulated Kinase protein also may interfere with factors that normally do not interact with the endogenous ERECTA, we believe that the observed dominant-negative interference is highly specific for the following reasons. First, the growth defects conferred by Kinase resemble or exaggerate the phenotypes of the erecta mutations, both in overall plant morphology and in underlying cellular defects (Figures 1, 3, and 8). Second, the ERECTA cis regulatory sequences that we used to express Kinase contain information sufficient for the proper expression of ERECTA, because a full-length ERECTA clone with these regulatory sequences fully complements erecta mutants (K.U. Torii, unpublished data). Therefore, neomorphic effects of Kinase in different tissue/cell types should be minimized. Third, Kinase did not inhibit the CLV LRR-RLK signaling pathway, which operates in the same cells that express ERECTA within the shoot and flower meristems (Figure 5). Fourth, introduction of a point mutation in the LRR domain of Kinase abolished the dominant negative effects without affecting the stability of the transcripts/proteins (Figure 6). This finding suggests that proper interaction with ligands and/or receptor partners that normally associate with native ERECTA likely are required for the observed dominant-negative interference, rather than the 100-fold accumulation of the protein causing some cellular toxicity.
The notion that having truncated ERECTA protein is worse than having none at all for Arabidopsis organ and internodal growth suggests complex redundancy in the signaling pathways involving ERECTA. One possible model is that several RLKs are capable of perceiving the same signal as ERECTA and of regulating partially overlapping pathways (Figure 9). Kinase may take up and deplete ligands and/or receptor partners of ERECTA that are shared by other RLKs and thus shut down whole pathways, which could operate in either a parallel or a convergent manner (Figure 9). The fact that a functional LRR domain is required for dominant-negative interference supports this hypothesis (Figure 6).
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Figure 9. Hypothetical Model for the ERECTA Signaling Pathway and Kinase Action.
(A) In wild-type plants, ERECTA associates with multiple partners (orange). Additional RLKs (blue) may share ligands (red) as well as receptor partners of ERECTA. The activation of these pathways promotes internode/organ elongation.
(B) erecta-105 plants do not have any ERECT. However, the receptor complex with overlapping function partially promotes internode/organ elongation.
(C) In ERECTA::Kinase/erecta-105 plants, highly stable ERECTA Kinase fragments shut down the entire pathway, presumably by forming inactive receptor heterodimers and/or depleting ligands for the RLK that has a function partially redundant with that of ERECTA.
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Such intricacy in signal transduction is well known in numerous animal receptor kinases. For example, mammalian platelet-derived growth factor (PDGF) exists as a homodimer and as heterodimers of three homologous polypeptides: PDGF A, B, and C (such as AA, AB, and BB) (Ataliotis and Mercola, 1997; Li et al., 2000). Two PDGF RTKs, PDGF R and PDGFR, recognize different PDGF isoforms with distinct affinity (Ataliotis and Mercola, 1997). This complex ligand receptor recognition property provides overlapping yet unique functions for PDGF signaling during mammalian embryogenesis. Consistently, expression of the dominant-negative Kinase form of PDGFR suppressed diverse signal transduction pathways mediated by multiple PDGF isoforms (Ueno et al., 1993). Similar complexity is documented for fibroblast growth factor RTK signaling pathways (Givol and Yayon, 1992).
The formation of an 400-kD Kinase protein complex is consistent with recent reports that plant LRR receptors constitute membrane-associated complexes (Trotochaud et al., 1999; Rivas et al., 2002a, 2002b). However, the components of the receptor complex could be distinct among CLV1, Cfs, and ERECTA. For instance, although active CLV1 complex contains KAPP and ROP small GTPase, neither is in the Cf4 or Cf9 complex (Trotochaud et al., 1999; Rivas et al., 2002a, 2002b). We found that ERECTA does not associate with KAPP (Figure 5C). Because the Kinase complex is nonfunctional, it also is unlikely to contain cytoplasmic factors that are recruited to the complex in a phosphorylation-dependent manner, such as ROP (Trotochaud et al., 1999). On the other hand, by analogy to the animal dominant-negative receptor kinases, the Kinase complex likely contains ligands and receptor partners for ERECTA and related RLKs (Figure 9). This notion is consistent with our finding that the point mutation within the LRR domain disrupts dominant-negative interference (Figure 6).
No matter how carefully our experiments were designed, we could not completely eliminate the possibility of nonspecific, neomorphic effects by Kinase. For instance, 100-fold accumulation of Kinase could titrate ligands for RLKs that have functions distinct from those of ERECTA, even if these ligands possess lower, and biologically irrelevant, affinity to the LRR domain of ERECTA. Another possibility is that the presence of Kinase alters the cellular equilibrium of cytoplasmic proteins involved in the regulation of multiple RLKs with distinct, unrelated functions. The availability of such proteins may decrease if, in wild-type plants, they interact with the ERECTA complex and then are recycled. KAPP is known to associate with a range of LRR-RLKs (Braun et al., 1997). Although we have shown that KAPP does not interact with ERECTA (Figure 5C), it might interact with a hypothetical ERECTA partner. Other unidentified downstream components of multiple RLKs also might be affected by Kinase. In any event, purification of the highly stable Kinase complex and subsequent isolation of the components of the ERECTA signaling pathway, as well as identification of the RLKs that possess redundant functions with ERECTA, will provide a complete picture of the biochemical mechanism of dominant-negative interference.
Role for the ERECTA Signaling Pathway in Organ Growth
Our observations revealed that erecta confers greatly increased cell size, primarily in the cortex, despite the fact that overall organ elongation is inhibited strongly (Figures 1 and 6). This finding is in contrast to what is known about almost all dwarf mutants described to date, which have reduced cell size, including those defective in biosynthesis and/or the perception of hormones such as auxins, gibberellins, and BRs (Timpte et al., 1992; Szekeres et al., 1996; Azpiroz et al., 1998; Fridborg et al., 1999), and indicates that ERECTA does not promote the general cell elongation process. The cellular defects in erecta are rather similar to the inhibition of cell cycle progression, which leads to cell enlargement even though overall plant size is reduced (Wang et al., 2000; De Veylder et al., 2001). One possible explanation for these findings is that ERECTA is required for proper cell cycle progression. Consistent with this hypothesis, the ratio of 4C cells increased in both erecta and Kinase plants (Figures 8E and 8F). Perhaps in the absence of the ERECTA signal, the cortex cells in pedicels may not enter mitosis and instead undergo premature differentiation at the G2 stage. Detailed analysis of cell division patterns during pedicel development is required to test this hypothesis. In contrast to the findings in erecta, overexpression of the cyclin kinase inhibitors (ICKs/KRPs) in Arabidopsis reduced the ratio of 4C cells in the leaf as a result of slowed cell cycle progression and reduced endoreduplication (De Veylder et al., 2001). Therefore, ERECTA signaling and ICKs/KRPs may act on distinct aspects of cell proliferation.
The striking cellular phenotype of the Kinase/erecta-105 pedicels is the loss of organized cortex cell size and shape (Figure 8D), suggesting that a loss of the entire pathway not only inhibits cell proliferation but also disrupts the uniformity of cell proliferation. Perhaps ERECTA and overlapping signaling pathways provide positional cues for coordinated proliferation among cells of the same type and such coordination is essential for proper organ elongation. Identification of the ligands and receptor partners of ERECTA, and analysis of their tissue- and/or cell-specific localization, will reveal the source of the signals and their target cells and will provide a clear picture of ERECTA LRR-RLK signaling in organogenesis.
Kinase as a Potential Tool to Dissect RLK Signal Transduction in Plants
The dominant-negative approach using truncated receptor kinases has been instrumental in dissecting signal transduction pathways and elucidating the in vivo functions of animal receptor kinases. For example, microinjection of the Kinase forms of the fibroblast growth factor RTK and the activin (transforming growth factor ) receptor Ser/Thr kinase revealed their essential roles in mesoderm formation during Xenopus embryogenesis (Amaya et al., 1991; Hemmati-Brivanlou and Melton, 1992). In those experiments, microinjection of mutant mRNA up to 100-fold the normal level successfully led to a targeted inactivation of specific receptor kinases. Expression of the Kinase epidermal growth factor RTK in Drosophila eyes revealed that this RTK is required for the differentiation of all cell types (Freeman, 1996). By contrast, such an approach has not been exploited widely in plants because of a few cases of cosuppression (Conner et al., 1997; Schumacher and Chory, 2000) or the inability to detect the accumulation of the transgene products (He et al., 1998). Our study demonstrated the effectiveness of the dominant-negative approach in understanding the modes of action and redundancy in the ERECTA signaling pathway.
Genetic dissection of the RLK signaling pathways that regulate plant development has been successful when the phenotypic consequences of the loss of the ligand and the receptor were nearly identical. For instance, mutations in both CLV1 and CLV3, which encode an LRR-RLK and its corresponding ligand, respectively, confer identical phenotypes in meristem enlargement and increased floral organ numbers (Clark et al., 1993, 1995, 1997; Fletcher et al., 1999). Another example is the BR receptor BRI1. bri1 was identified as a BR-insensitive mutant that displays characteristic BR-deficient phenotypes (e.g., "cabbage"-like rosettes and seedling deetiolation) (Clouse et al., 1996; Kauschmann et al., 1996; Li and Chory, 1997).
Despite the fact that RLKs constitute a large family in higher plants, with >600 members in Arabidopsis, only a handful of RLKs are known to have defined biological functions, and the identity of the corresponding ligands is far less clear (Torii, 2000; Shiu and Bleecker, 2001). It is not yet understood whether a majority of other RLKs possess modes of action similar to that of ERECTA or whether their Kinase fragments retain partial activity to transduce signals. In any event, the truncated receptor kinase system established in this study could serve as a powerful, alternative tool to elucidate the biological functions and signaling components of other RLKs, whose single loss-of-function mutations may not give any phenotypes as a result of complex redundancy.
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METHODS
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Plant Materials and Growth Conditions
Arabidopsis thaliana ecotype Columbia was used as the wild type. erecta-103 and erecta-105 were backcrossed four times into the wild type before use (Torii et al., 1996). Plants were grown on soil mixture (Sunshine Mix4:vermiculite:perlite, 2:1:1, Sun Gro Horticulture Canada, Seba Beach, Canada) supplemented with 0.85 mg/cm3 Osmocoat 14-14-14 (Scotts, Maysville, OH) under an 18-h-light/6-h-dark cycle at 21°C.
Generation of Transgenic Plants Expressing Kinase
To construct the plasmid carrying the truncated ERECTA, we introduced the stop codon behind the putative transmembrane domain at amino acid position 615 by PCR. PCR was performed using pKUT196, which contains the entire ERECTA locus from Columbia, as a template and primers Erg5858link (5'-ATGAATTCTGTCTGCAGTGTCAATCTCTA-3') and ER6000Bam.rc (5'-TCAGGATCCTATGATCCATCAAGAAAAGGA-GG-3'). The amplified fragment was digested with PstI and BamHI and introduced into plasmid pKUT197 to generate pESH101. The EcoRI-BamHI fragment of pESH101, which contains the 1.7-kb ERECTA promoter and the coding region of the truncated ERECTA, was cloned into pKUT531, a pPZP222-based binary vector, which contains the 1.9-kb ERECTA terminator (Hajdukiewicz et al., 1994). The plasmid was named pESH201.
To generate the Kinase–c-Myc construct, the following cloning steps were performed. To introduce the BamHI site after Ser-615 of ERECTA, PCR was performed using pKUT196 as a template and primers Erg5868link (5'-ATGAATTCTGTCTGCAGTGTCAATCTCTA-3') and ER-6000link.rc (5'-TCAGGATCCGCTGATCCATCAAGAAAAGGAGG-3'). The amplified fragment was cloned into PstI-BamHI–digested pKUT197 to generate pESH113. The triple c-Myc sequence was amplified by PCR using primers myc-5 (5'-GAAGATCTCGAGTTCGGTGAACAAAAGTT-3') and myc-3 (5'-CGGGATCCTTACCCTAGCTTTCCGTTCAAGT-3') with pSLJ13471 as a template (Jones et al., 1992). The amplified fragment was digested with BglII and BamHI and inserted into BamHI-digested pESH113. The resulting plasmid, pESH115, contains the additional sequence 5'-ADLEFG(EQKLISEEDLNG)3KLG-3' after Ser-615 of ERECTA. EcoRI-BamHI–digested pESH115 was cloned into pKUT531 to generate pESH215.
To generate KinaseM282I, PCR was performed using erecta-103 genomic DNA as a template with primers ERg1761 (5'-GTATATCTA-AAAACGCAGTCG-3') and ERg2339rc (5'-CAACAACATTGAAGGTGACATTTT-3'). The amplified fragment was digested with SpeI and SacI and replaced the SpeI-SacI fragment of pESH101. The resulting plasmid, pKUT572.3, was digested with AflII and SacI and inserted into pESH201 to generate pKUT574.3. The sequences of all fragments created by PCR were confirmed. pESH201, pESH215, and pKUT574.3 were introduced into Agrobacterium tumefaciens strain GV3101/pMP90 by electroporation and into Arabidopsis wild-type and erecta-105 plants using the vacuum infiltration method (Bechtold et al., 1993).
Scanning Electron Microscopy
Tissue samples were fixed overnight in 4% (v/v) glutaraldehyde in 25 mM NaPO4 buffer, pH 7.0, and subsequently with 1% osmium tetroxide in 25 mM NaPO4 buffer for 4 to 5 days at 4°C. The samples were dehydrated with a graded series of ethanol, critical point dried, sputter coated with gold, and observed with a scanning electron microscope (JEOL 840A).
Light Microscopy
Tissue samples were fixed overnight in 4% (v/v) paraformaldehyde in 25 mM NaPO4 buffer, pH 7.0, at 4°C, dehydrated with a graded series of ethanol, and infiltrated with polymethacryl resin Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany) followed by embedding and polymerization in Technovit 7100. Nine-micrometer sections were prepared using a Leica RM-6145 microtome (Wetzlar, Germany). The tissue sections were stained with 0.1% toluidine blue in 0.1 M NaPO4 buffer, pH 7.0, and observed under bright-field illumination.
Antiserum Production and Purification
The cDNA sequence encoding the extracellular domain of ERECTA (ERLRR; amino acids 36 to 577) was amplified using primers ERLRRab5 (5'-CGGAATTCTCATTCAAAGATGTGAACAATG-3') and ERLRRab3 (5'-CGTCTAGACTATGACACTCGTACAGTTCGA-3'), with pKUT161 as a template (Torii et al., 1996). The amplified fragment was inserted in the EcoRI-XbaI–digested modified pSP73AatII vector (Promega) to generate pKUT534. The sequence was confirmed. Subsequently, the fragment was inserted in pMal-c2 vector (New England Biolabs, Beverly, MA) and pGEX4T-1 vector (Amersham Pharmacia Biotech) to generate pKUT535 (maltose binding protein [MBP]-ERLRR) and pKUT538 (glutathione S-transferase [GST]-ERLRR), respectively. The fusion proteins were expressed in Escherichia coli BL21/DE3(pLysS). The inclusion bodies of E. coli expressing MBP-ERLRR were separated by SDS-PAGE, and the recombinant protein was excised from the gel. Polyclonal ERLRR antisera were raised in rabbits at Cocalico Biologicals (Reamstown, PA). Affinity purification of antibodies was performed using the GST-ERLRR fusion protein immobilized on nitrocellulose membranes.
Protein Gel Blot and Immunoblot Analyses
One gram of Arabidopsis bud clusters (inflorescence tips) was ground in liquid nitrogen, mixed with 2 mL of ice-cold lysis buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 100 mM NaCl, 0.1% SDS, 0.1% Triton X-100, 0.7% -mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride) and 0.5 mL of loading buffer (200 mM Tris-HCl, pH 6.8, 8% SDS, 0.4% bromphenol blue, 40% glycerol, and 10% -mercaptoethanol), and boiled for 5 min. Proteins were separated by 8% SDS-PAGE. SeeBlue prestained protein standards (Invitrogen, Carlsbad, CA) were used as molecular mass markers. For visualization of the total proteins, the gel was stained with Coomassie Brilliant Blue R250. For immunoblot analysis, the proteins were transferred from the gel to Hybond enhanced chemiluminescence nitrocellulose membranes (Amersham Pharmacia Biotech) using a semidry blotting apparatus (Owl Separation Systems, Portsmouth, NH). The membranes were blocked with 5% BSA in PBS for 2 h at room temperature and probed with primary antibody at a dilution of 1:15 for the affinity-purified ERLRR polyclonal antibody or 1:600 for the 9E10 anti-c-Myc monoclonal antibody (Covance, Richmond, CA) in PBS with 1% BSA at 4°C overnight. Goat anti-rabbit and sheep anti-mouse horseradish peroxidase–linked antibodies were used as secondary anti-ERLRR and anti-c-Myc antibodies, respectively, at a dilution of 1:35,000 in 0.1% Tween 20/PBS for 1 h at room temperature. The detection of ERECTA, Kinase, and Kinase–c-Myc was performed with the Chemiluminescence assay kit (Amersham Pharmacia Biotech).
Reverse Transcriptase–Mediated PCR
Total RNA was isolated from Arabidopsis bud clusters using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and treated with DNaseI Amp grade (Gibco BRL). First-strand cDNA was synthesized from 2 µg of RNA with random hexamer primers using the ThermoScript reverse transcriptase–mediated PCR system (Gibco BRL) according to the manufacturer's instructions. PCR was performed with 0.5 µL of the first-strand reaction at 96°C for 2 min, then with varying numbers of cycles at 96°C for 35 s, 60°C for 45 s, 72°C for 90 s, and then at 72°C for 10 min. The primers ERK7 (5'-CACAGAGACGTGAAGTCGT-3') and ERg7361rc (5'-AGC-TTAACGCAACGAAAAGATACC-3') were used to amplify endogenous ERECTA. The primers ERg5022 (5'-CTTGAGTAGAAATCATATAACT-3') and ERg5757rc (5'-TGACACGGTGAGTTTAGCCAA-3') were used to simultaneously amplify both the endogenous ERECTA and the introduced Kinase. The primers ERg5022 (5'-CTTGAGTAGAAATCATATAACT-3') and ERg7361.rc (5'-AGCTTAACGCAACGAAAAGATACC-3') were used to amplify the introduced Kinase. Transcripts of the actin gene were amplified as a control using primers ACT2-1 (5'-GCCATCCAAGCTGTT-CTCTC-3') and ACT2-2 (5'-GCTCGTAGTCAACAGCAACAA-3'). WUS transcripts were amplified as described by Hamada et al. (2000). Reverse transcriptase–mediated PCR products were electrophoresed on agarose gels and visualized by staining with ethidium bromide.
Far-Western Analysis of the ERECTA–KAPP Interaction
For protein-protein interaction (far-western) blot analysis, the kinase domains of ERECTA and RLK5 were expressed as MBP fusions. The ERECTA kinase domain (corresponding to amino acids 611 to 977) was amplified from cDNA using the primers ERK4 (5'-CGGAATTCACTAGTA-CCATGGACAAACCAGTAACTTATTCG-3') and ER3rc (5'-CGGGATCCA |
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Abstract
INTRODUCTION
