Distinct signaling mechanisms in multiple developmental pathways by the SCRAMBLED receptor of Arabidopsis.

Distinct Signaling Mechanisms in Multiple Developmental Pathways by the SCRAMBLED Receptor of Arabidopsis1[OPEN] Su-Hwan Kwak*, Sooah Woo, Myeong Min Lee, and John Schiefelbein Biology Department, Long Island University, Brooklyn, New York 11201 (S.-H.K.); Department of Systems Biology, Yonsei University, Seoul 120–749, Korea (S.W., M.M.L.); and Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109 (J.S.)

SCRAMBLED (SCM), a leucine-rich repeat receptor-like kinase in Arabidopsis (Arabidopsis thaliana), is required for positional signaling in the root epidermis and for tissue/organ development in the shoot. To further understand SCM action, we generated a series of kinase domain variants and analyzed their ability to complement scm mutant defects. We found that the SCM kinase domain, but not kinase activity, is required for its role in root epidermal patterning, supporting the view that SCM is an atypical receptor kinase. We also describe a previously uncharacterized role for SCM in fruit dehiscence, because mature siliques from scm mutants fail to open properly. Interestingly, the kinase domain of SCM appears to be dispensable for this developmental process. Furthermore, we found that most of the SCM kinase domain mutations dramatically inhibit inflorescence development. Because this process is not affected in scm null mutants, it is likely that SCM acts redundantly to regulate inflorescence size. The importance of distinct kinase residues for these three developmental processes provides an explanation for the maintenance of the conserved kinase domain in the SCM protein, and it may generally explain its conservation in other atypical kinases. Furthermore, these results indicate that individual leucine-rich repeat receptor-like kinases may participate in multiple pathways using distinct signaling mechanisms to mediate diverse cellular communication events.

Members of the large leucine-rich repeat (LRR) receptorlike kinase (RLK) family participate in diverse cellular communication events in plants, including hormonal, developmental, and environmental signaling (Morillo and Tax, 2006; De Smet et al., 2009; Gish and Clark, 2011; Antolín-Llovera et al., 2012). The extracellular LRR domains are thought to interact with specific external factors, which trigger changes in the activity of the intracellular kinase domain to mediate appropriate molecular responses. Furthermore, in some instances, multiple LRR-RLKs with distinct and/or overlapping properties may participate in a common signaling pathway, providing the opportunity for redundancy or combinatorial action to increase the diversity of potential responses (Tӧr et al., 2009; Zhao, 2009). The root epidermal cells of Arabidopsis require SCRAMBLED (SCM), a member of the LRR-RLK family, 1 This work was supported by the U.S. National Science Foundation (grant no. IOS–1121602 to J.S. and S.-H.K.), the Next-Generation BioGreen21 Program of the Rural Development Administration of the Republic of Korea (grant no. PJ008207 to M.M.L.), and the faculty start-up fund from the Brooklyn Campus of Long Island University (to S.-H.K.). * Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Su-Hwan Kwak ([email protected]). [OPEN] Articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.114.247288

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to interpret their positions and adopt appropriate cell fates (Kwak et al., 2005). In Arabidopsis (Arabidopsis thaliana) roots, immature epidermal cells outside the cleft between two cortical cells (the H position) develop into root-hair cells, whereas epidermal cells outside a single cortical cell (the N position) adopt the nonhair cell fate (Schiefelbein et al., 2009; Grebe, 2012). Root epidermal cells in scm null mutants (e.g. scm-1 and scm-2) tend to adopt their cell fates in a position-independent manner, leading to an abnormal cell-type pattern (Kwak et al., 2005). SCM appears to act by repressing the expression of WEREWOLF, a nonhair cell transcription factor gene, resulting in its differential expression among epidermal cells at an early stage of development. This initial bias is magnified by feedback mechanisms involving CAPRICE, MYB23, and SCM itself that determine which epidermal cells ultimately express GLABRA2 (GL2), a homeodomain transcription factor gene that promotes nonhair cell differentiation (Kwak and Schiefelbein, 2007, 2008; Kang et al., 2009; Song et al., 2011; Bruex et al., 2012). SCM, also known as STRUBBELIG, regulates additional developmental processes in Arabidopsis including floral organ development, coordinated shoot cell division/enlargement (Chevalier et al., 2005), and the formation and venation of leaves (Lin et al., 2012). Interestingly, SCM appears to lack phosphotransfer activity by in vitro assays, and some SCM proteins harboring mutations in conserved kinase domain residues are able to rescue the flower organ defects in scm mutant plants (Chevalier et al., 2005; Vaddepalli et al., 2011). These findings suggest that kinase activity is not required

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Multiple SCRAMBLED Receptor Developmental Pathways

for the function of SCM in shoot organ/tissue development, although the importance of the kinase domain in root epidermal patterning has not been investigated. In this study, we sought to define the role of the SCM kinase domain in the root epidermis pathway by assessing the ability of altered SCM proteins to complement scm mutant phenotypes. We also uncovered two SCM-mediated developmental processes, involving fruit dehiscence and inflorescence outgrowth. Our comparative analysis of these three processes suggests that root epidermis patterning, fruit dehiscence, and inflorescence development possess distinct requirements for SCM kinase domain sequences. This indicates that SCM uses developmental pathway-specific mechanisms of receptor signaling to mediate diverse cellular communication events. RESULTS The SCM Kinase Domain, But Not Kinase Activity, Is Required for Root Epidermal Patterning

We first tested the kinase activity of the wild-type SCM protein by using an in vitro phosphorylation assay.

The kinase domains of the wild-type SCM protein and a mutant SCM protein with an amino acid substitution in the subdomain II (called SCM mKD1, K525E; Fig. 1) were expressed and purified from Escherichia coli. The kinase domain of Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) was also included as a control. Consistent with a prior report (Chevalier et al., 2005), we observed no detectable autophosphorylation activity from the kinase domain of the wild-type SCM. As expected, the kinase domain containing the K525 substitution also showed no detectable autophosphorylation activity (Fig. 1C). Given the lack of SCM kinase activity, we wanted to test the importance of the SCM kinase domain for SCM function, by assessing the effect of point mutations and deletions in conserved regions of the SCM kinase domain. Three point mutants (mKD1, mKD2, and mKD3) were generated in genomic sequences, fused with GFP, and expressed in the scm-2 mutant under the control of the native SCM promoter (Fig. 1A). In previous work, this promoter region was shown to be capable of driving expression of wild-type SCM to fully complement the

Figure 1. Structure of SCM fusion proteins and sequence of SCM kinase domain. A, Structure of SCM-GFP fusion proteins tested for complementation of the scm-2 mutant in this study. Major protein domains, from the N terminus (left) to C terminus (right), include the six predicted LRRs (gray), transmembrane domain (black), juxtamembrane domain (black line), kinase domain, and GFP sequence. Amino acid substitutions generated in the SCM kinase domain and their equivalent mutant alleles in other Arabidopsis LRR-RLKs are indicated. B, Amino acid alignment of the kinase domains of the LRR-RLKs of SCM, CLV1, ERECTA, BAK1, BRI1, and FLS2. The conserved subdomains of kinases are marked with numbers from I to XI. Black circles and squares represent amino acid changes present in aphenotypic alleles and phenotypic alleles, respectively, of SCM mutants (Chevalier et al., 2005; Vaddepalli et al., 2011). Asterisks mark three amino acids in subdomains II, VIb, and VII, which are altered in alternative catalytic function kinases (Dardick and Ronald, 2006). Predicted a-helices and b-sheet structures are shown under the amino acid sequences (from the Phyre2 server, http://www.sbg.bio.ic.ac.uk/~phyre2; Kelley and Sternberg, 2009). C, In vitro autophosphorylation analysis of the SCM kinase domain. The purified proteins from an E. coli extract were separated and stained with Coomassie Brilliant Blue (left) and autoradiography was obtained (right). Arrowheads represent the purified SCM or AtSERK1 kinase domain. AtSERK1 KD, AtSERK1 kinase domain; BAK1, BRI1-ASSOCIATED RECEPTOR KINASE1; KD, kinase domain; SCM KD, SCM kinase domain; SCM mKD1, SCM kinase domain with K525E substitution; SM, size marker. Plant Physiol. Vol. 166, 2014


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scm-2 root mutant phenotype (Kwak et al., 2005; Kwak and Schiefelbein, 2008). After transformation of each mutant SCM construct into the scm-2 mutant, we confirmed that each of the GFP-tagged proteins was expressed and was localized appropriately to the plasma membrane of root epidermal cells using confocal microscopy (Fig. 2, G–K). Furthermore, reverse transcription (RT)-PCR experiments were conducted using primers specific for mGFP5 to confirm expression of each fusion gene in shoots (Fig. 2L). In the mKD1GFP protein, Glu replaces Lys-525 (K525E; Fig. 1, A and B). Lys-525 is an invariant residue in the

kinase subdomain II that is implicated in transferring phosphate to substrates (Hanks et al., 1988). Replacement of this Lys with Glu in other LRR-RLKs, such as BRASSINOSTEROID INSENSITIVE1 (BRI1), CLAVATA1 (CLV1), and SERK1, abolished their autophosphorylation activity (Stone et al., 1998; Oh et al., 2000; Shah et al., 2001). However, our experiments indicate that the mKD1GFP protein is capable of functioning in epidermal patterning (Fig. 2C; Table I). In wild-type Arabidopsis roots, epidermal cells in the H position (cell files marked with asterisks in Fig. 2) lack expression of the nonhair marker GL2::GUS, whereas cells in the N position express

Figure 2. Root epidermal cell pattern in scm-2 mutants bearing SCM-GFP fusions with mutations in the kinase domain and localization of SCM-GFP fusion proteins in roots. A to F, Expression pattern of the nonhair cell marker GL2::GUS in the root epidermal cells from the meristematic region of wild-type (A), scm-2 (B), scm-2 SCM::mKD1GFP (C), scm-2 SCM::mKD2GFP (D), scm-2 SCM::mKD3GFP (E), and scm-2 SCM::dKD2GFP (F) roots. Asterisks indicate cell files in the H position, which normally lack GL2::GUS expression. G to K, Confocal microscopy images, focused on the developing epidermis, were obtained from 4-d-old scm-2 roots expressing intact SCMGFP (G), mKD1GFP (H), mKD2GFP (I), mKD3GFP (J), and dKD2GFP (K) fusion proteins under control of the SCM regulatory regions. Green fluorescence indicates SCM-GFP fusion proteins and red indicates cell walls (propidium iodide staining). (L) RT-PCR was used to confirm expression of mutated SCM-GFP constructs in the shoots of 6-d-old seedlings. Total RNA from scm-2 (1), scm-2 SCM::mKD1GFP (2), scm-2 SCM::mKD2GFP (3), scm-2 SCM::mKD3GFP (4), and scm-2 SCM::dKD2GFP (5) was used in RT-PCR with mGFP5 primers (to detect transgene expression) and EF1a primers (control). WT, Wild type. Bar = 50 mm. 978

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GL2::GUS, providing a simple assay for root epidermal patterning (Fig. 2A). In the scm-2 mutant, the GL2:: GUS-expressing and GL2::GUS-nonexpressing cells are not limited to specific cell files, because positiondependent cell fate patterning is disrupted (Fig. 2B). We found that the SCM::mKD1GFP transgene largely restored epidermal patterning in the scm-2 roots (Fig. 2C; Table I), suggesting that SCM kinase activity is not required for normal root epidermal patterning, and consistent with an independent report using a K525E SCM mutant that retains normal function in shoot development (Chevalier et al., 2005). The mKD2GFP protein has an amino acid substitution at Glu-686 to Lys (E686K) in the kinase subdomain IX (Fig. 1, A and B). The bri1-101 allele has the same amino acid change, and this alteration abolishes BRI1 kinase activity (Friedrichsen et al., 2000; Nam and Li, 2002). However, we found that the SCM::mKD2GFP construct was able to rescue the defect in root epidermal patterning in the scm-2 mutant plants although cells in the N position showed a marginal defect (Fig. 2D; Table I). Like the results with mKD1GFP (above), these data further support the view that kinase activity is not required for SCM to function sufficiently in root epidermal patterning. In the mKD3GFP protein, the conserved Gly-681 in the kinase subdomain IX is changed to Arg (G681R; Fig. 1, A and B). The clv1-2 and flagellin-sensitive2-17 (fls2-17) alleles have the analogous mutation in their kinase subdomain IX. The clv1-2 mutation causes an intermediate phenotypic effect, and the clv1-2 protein (expressed in E. coli) retains some autophosphorylation activity (Diévart et al., 2003). The fls2-17, an allele of the LRR-RLK FLAGELLIN-SENSITIVE2 (FLS2), lacked autophosphorylation activity (Gómez-Gómez et al., 2001). In our molecular complementation test, the SCM:: mKD3GFP transgene failed to restore the normal root epidermal pattern in the scm-2 mutant (Fig. 2E; Table I). This result suggests that certain residues in the SCM kinase domain may be important for proper SCM function. Endoplasmic reticulum retention-like localization of GFP was detected in the scm-2 mKD3GFP root epidermal cells (Fig. 2J). We cannot rule out the possibility that the endoplasmic reticulum retention of mKD3GFP proteins

contributed to failure in complementation of root epidermal patterning of the scm-2 mutant; however, we could clearly detect localization of mKD3GFP fluorescence in the plasma membrane as well (Fig. 2J). To further test the importance of the SCM kinase domain, we analyzed the effect of a mutant SCM protein lacking the entire cytoplasmic kinase domain but retaining the juxtamembrane domain (dKD2GFP; Fig. 1A). The scm-2 plants bearing this construct (SCM::dKD2GFP) exhibited an abnormal root epidermal pattern, resembling the scm-2 mutant (Fig. 2F; Table I). Thus, we conclude that although kinase activity is not required, the kinase domain of SCM is needed for it to mediate root epidermal cell patterning.

SCM Affects Fruit Dehiscence in a Largely Kinase-Independent Manner

During the course of this study, we discovered a previously unreported defect in fruit dehiscence in the scm-2 mutant. In contrast with the wild type, the scm-2 siliques failed to open in mature dry plants (Fig. 3, A and B). A similar phenotype was observed in the strong scm-1 mutant, but not in the weaker scm-3 mutant (data not shown). Silique length was similar in wild-type and scm-2 mutant plants, and the scm-2 siliques possessed normal-appearing valves and replum (Fig. 3, A and B), suggesting that the dehiscence defect was not attributable to a major structural defect. Furthermore, mature viable seeds were produced by scm-2 and although they were not released spontaneously from mature siliques, they could be released upon mechanical pressure. Using a SCM::GUS transcriptional reporter line, we observed GUS expression between the valves of the developing siliques in the area including the developing replum (see Fig. 5P). These results suggest that SCM signaling may be required for proper development of the tissues required for fruit pod opening where valves and replum meet. It is known that scm mutants possess twisted stems, floral organs, and siliques (Chevalier et al., 2005). In our analysis of scm-2 siliques, we found that 66% exhibit a

Table I. Cell-type specification in the root epidermis of wild-type, scm-2, and transgenic lines Values indicate the mean 6

SD

of three counting sessions. Epidermal cells in at least 14 roots were counted for each counting session. Cells in the H Position

Line

GL2::GUS- Nonexpressing Cells

Wild type (Columbia) scm-2 scm-2 mKD1GFP scm-2 mKD2GFP scm-2 mKD3GFP scm-2 dKD2GFP/+b dKD2GFP

92.7 65.4 91.4 96.6 66.1 71.5 96.8

6 6 6 6 6 6 6

1.2 5.1 2.4 2.2 1.5 6.8 1.1

Cells in the N Position

GL2::GUS- Expressing Cells

7.3 34.6 8.6 3.4 33.9 28.5 3.2

6 6 6 6 6 6 6

1.2 5.1a 2.4 2.2 1.5a 6.8a 1.1

GL2::GUS-Nonexpressing Cells

0.2 24.9 8.7 1.7 19.3 25.8 1.5

6 6 6 6 6 6 6

0.3 4.7a 4.6 0.6a 1.1a 6.7a 0.3a

GL2::GUS- Expressing Cells

99.8 75.1 91.3 98.3 80.7 74.2 98.5

6 6 6 6 6 6 6

0.3 4.7 4.6 0.6 1.1 6.7 0.3

a b Significant difference from the Columbia wild-type control (P , 0.05, Student’s t test). A segregating pool from a scm-2/scm-2 dKD2GFP/+ parent was used for counting. None of the individual roots used for counting showed complementation of the epidermal pattern defect of the scm-2 mutant (n = 42).



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Figure 3. Fruit (silique) dehiscence in scm-2 mutants bearing SCM-GFP fusions with mutations in the kinase domain. A to F, Mature siliques illustrating valve separation phenotypes in wildtype (A), scm-2 (B), scm-2 SCM::mKD1GFP (C), scm-2 SCM::mKD2GFP (D), scm-2 SCM:: mKD3GFP (E), and scm-2 SCM::dKD2GFP (F) plants. G, Quantification of silique dehiscence phenotype. Dehiscing siliques and nondehiscing siliques were counted from five different plants of each genetic line. Error bars represent the SD. Single asterisks indicate a significant difference from the Columbia wild-type control (Col WT; P , 0.05, Student’s t test). The double asterisk indicates a significant difference from both the wild-type control and the scm-2 mutant (P , 0.05, Student’s t test). Bar = 2 mm.

strong twisted phenotype, 26% possess a mild twisted phenotype (slightly tilted valve margin relative to the silique axis), and 8% exhibit no twisting (i.e. normal structure; n = 635). However, all of these siliques failed to open spontaneously upon maturation, showing that twisting per se is not responsible for the silique dehiscence defect. We examined scm-2 plants bearing the SCM::mKD1GFP, SCM::mKD2GFP, SCM::mKD3GFP, or SCM::dKD2 transgenes to evaluate the importance of the SCM kinase domain for SCM’s role in the fruit dehiscence pathway. The siliques of scm-2 plants containing either the SCM:: mKD1GFP or SCM::mKD2GFP transgenes exhibited normal development and spontaneous opening at maturity (Fig. 3, C, D, and G), indicating that SCM kinase activity is not required for SCM to function in this pathway. However, the scm-2 SCM::mKD3GFP plants exhibited the fruit dehiscence defect (Fig. 3, E and G), implying that the conserved Gly-681 is important for this process. Interestingly, the kinase domain deletion variant (dKD2GFP and dKD2) was able to partially rescue the silique opening abnormality in the scm-2 mutant (Figs. 3, F and G, and 5N), which indicates that the SCM kinase domain is not essential for SCM to participate in fruit 980

dehiscence. Furthermore, this property distinguishes SCM’s role in fruit dehiscence from its role in root epidermal patterning. We confirmed the localization of the SCM-GFP fusion proteins in cells of the upper replum of mature flowers by confocal microscopy. The GFP fluorescence was detected in the plasma membrane of the scm-2 SCMGFP, scm-2 mKD1GFP, and scm-2 mKD3GFP plants although mKD3GFP failed to complement the silique dehiscing phenotype of scm-2 (Fig. 4).

SCM Is Involved in Inflorescence Size Regulation

While analyzing the effect of the mutant SCM proteins, we discovered that plants from the scm-2 SCM:: mKD2GFP, scm-2 SCM::mKD3GFP, and scm-2 SCM:: dKD2GFP lines exhibit a dramatic reduction in inflorescence size. Rather than the normal-sized inflorescences found in the wild-type, scm-2, and scm-2 SCM::mKD1GFP plants, these plants produced extremely small inflorescence outgrowths from normal-sized rosettes (Fig. 5, A–F and S). Despite this reduction in inflorescence size, relatively normal flowers and floral organs are formed (Fig. 5, L and O), suggesting that cell and organ formation Plant Physiol. Vol. 166, 2014



occur successfully at the inflorescence meristem. Furthermore, we observed no significant difference in flowering time between plants expressing the dKD2GFP protein and nontransgenic wild-type plants (mean number 6 standard deviation of rosette leaves at floral transition: 16.0 6 3.7 [n = 29] and 16.2 6 3.4 [n = 30], respectively), indicating that dKD2GFP does not alter floral induction. Because the scm-2 SCM::mKD2GFP, scm-2 SCM:: mKD3GFP, and scm-2 SCM::dKD2GFP lines exhibited the same phenotype, we conducted a detailed genetic analysis on only one of these lines, scm-2 SCM::dKD2GFP. First, we assessed the possibility of a dosage effect, by analyzing scm-2/scm-2 SCM::dKD2GFP/+ T1 plants. These plants exhibited the same inflorescence size defect, indicating that a single copy of SCM::dKD2GFP is sufficient to generate the abnormal phenotype. Consistent with this, after the self-pollination of T1 plants, the inflorescence size phenotype segregated at a ratio of approximately 3:1 (defective versus normal inflorescence growth). Suspecting a dominant negative effect, we generated and analyzed various combinations of wild-type SCM and SCM::dKD2GFP genes. By genotyping the segregating F2 progeny derived from a cross of wild-type and scm-2 SCM::dKD2GFP plants, we found that homozygous SCM::dKD2GFP/SCM::dKD2GFP plants exhibited the inflorescence size defect, no matter their SCM genotype

(Fig. 5, I, K, and L). By contrast, a reduction in the dosage of SCM::dKD2GFP in either a homozygous or heterozygous SCM background (SCM/SCM SCM:: dKD2GFP/+ or SCM/scm-2 SCM::dKD2GFP/+) generated plants with normal-sized inflorescence (Fig. 5, H and J). As a further test, we crossed scm-2 SCM::mKD1GFP (normal inflorescence size) with scm-2 SCM::dKD2GFP plants (abnormal inflorescence size) and found that F1 offspring produced a normal-sized inflorescence (Fig. 5G). Together, these results are consistent with the possibility that SCM::dKD2GFP interferes with the normal role of SCM in inflorescence development during the floral transition. In support of this, a high level of SCM expression is detected in the inflorescence meristem by in situ hybridization (Chevalier et al., 2005), and the Electronic Fluorescent Pictograph browser shows a higher level of SCM expression in the inflorescence meristem than the vegetative shoot apical meristem (Winter et al., 2007). To determine whether the GFP portion of the dKD2GFP fusion protein might be responsible for the inflorescence defect, we generated plants expressing the dKD2 protein alone under the control of the SCM promoter. We found that these SCM::dKD2 plants also produced an extremely small inflorescence in both wild-type and scm-2 backgrounds (Fig. 5, M and N), suggesting that the GFP is not required for this effect. Finally, we generated a mutant SCM construct that encodes a protein

Figure 4. Expression and localization of intact and mutated SCM-GFP fusion proteins in scm-2 replum. Confocal microscopy images, focused on the epidermis, were obtained from the replum of 3-week-old scm-2 flowers expressing intact SCMGFP (A–C), mKD1GFP (D–F), and mKD3GFP (G–I) fusion proteins under control of the SCM regulatory regions. Green fluorescence indicates SCM-GFP fusion proteins (A, D, and G), and red indicates autofluorescence (B, E, and H). The merge of both fluorescences is shown in C, F, and I. Bar = 20 mm.



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Figure 5. Inflorescence production in plants expressing SCM-GFP fusion proteins with mutations in the kinase domain. A, SCM/ SCM (the wild type). B, scm-2/scm-2. C, scm-2/scm-2 SCM::mKD1GFP/SCM::mKD1GFP. D, scm-2/scm-2 SCM::mKD2GFP/ 982




containing the extracellular domain, the transmembrane domain, and only a small portion (17 amino acids) of the juxtamembrane domain (called SCM::dKD1). The resulting expression of SCM::dKD1 in the wild type and the scm-2 background also exhibited the abnormal inflorescence phenotype (Fig. 5, Q and R). Quantification of the inflorescence size phenotype revealed a mild reduction in inflorescence stem length in the scm-2 mutant (Fig. 5S), rescue of the scm-2 defect by the mKD1GFP protein, and significant inhibition of inflorescence stem length by the mKD2GFP, mKD3GFP, and dKD2GFP proteins in the scm-2 background. The dKD2GFP protein repressed inflorescence stem growth in the wild-type background as well (Fig. 5S). Given the effect of mutant SCM proteins on inflorescence development, we assessed the expression and localization of the SCM-GFP fusion proteins in the inflorescence meristem of 3-week-old plants by confocal microscopy. We were able to detect GFP fluorescence in the plasma membrane of cell in the inflorescence meristem of scm-2 SCMGFP, scm-2 mKD1GFP, scm-2 mKD2GFP, scm-2 mKD3GFP, scm-2 dKD2GFP, and dKD2GFP plants (Fig. 6), implying that membrane-localized SCM proteins play a role in inflorescence development. Because the dKD2GFP protein appears to interfere with SCM’s role in inflorescence formation, we examined whether it may inhibit or interfere with SCM function in root epidermal patterning or in fruit dehiscence. To test this, we generated plants expressing dKD2GFP in a wild-type background. Although a slight defect was shown in N-position cells, these SCM::dKD2GFP/ SCM::dKD2GFP plants produced a near-normal pattern of root epidermal cells (Fig. 7, A–D; Table I) and normal silique opening at maturity (Fig. 7E), suggesting that the dKD2GFP protein does not interfere with these SCM pathways. This implies that the inhibitory effect of the SCM kinase mutants (mKD2GFP, mKD3GFP, dKD2GFP, dKD2, and dKD1) is largely limited to the inflorescence developmental pathway, distinguishing it from SCM’s action in root epidermis and fruit development DISCUSSION Two SCM-Associated Developmental Pathways in Arabidopsis

The SCM LRR-RLK of Arabidopsis was reported to participate in several developmental processes, including

root epidermal cell patterning, ovule formation, and shoot tissue morphogenesis (Chevalier et al., 2005; Kwak et al., 2005; Lin et al., 2012). Here we present evidence for two additional roles of the SCM receptor: fruit dehiscence and inflorescence development. Fruit opening (or dehiscence) in Arabidopsis involves the separation of the two valves (pod walls) at maturity to release seeds (Robles and Pelaz, 2005). Dehiscence depends upon proper tissue differentiation during fruit development to generate the valves, replum, and valve margins (which later become the dehiscence zone). Within the dehiscence zone, degradation of the middle lamella between cells of the separation layer leads to detachment of the valves from the replum. The fruit dehiscence defect in the scm mutants is not associated with abnormalities in silique structure, so it is likely attributable to failure of dehiscence zone differentiation or activity. A gene regulatory pathway controlling dehiscence zone development was defined, including the transcription factor genes INDEHISCENT, SHATTERPROOF1, SHATTERPROOF2, ALCATRAZ, FRUITFULL, and REPLUMLESS (Dinneny and Yanofsky, 2005; Robles and Pelaz, 2005). One exciting possibility is that SCM-mediated cell-cell signaling may establish appropriate expression domains for these dehiscence zone transcription factors during fruit development. Our results also indicate that SCM is involved in regulating inflorescence development after floral induction. Although Arabidopsis plants normally produce an extensive branching inflorescence, transgenic lines expressing kinase-domain mutant versions of SCM produce an extremely small inflorescence with relatively few branches and flowers. Because organogenesis and seed production still occur in these lines, we conclude that the primary effect is on control of inflorescence size. Furthermore, given that constructs generating this abnormal inflorescence phenotype act in a dosage-sensitive manner relative to the wild-type SCM gene, this defect is likely due to interference with the normal function of SCM during inflorescence development, possibly by the poisoning of signaling complexes by the kinasedomain mutant versions. The role of SCM in this process likely evaded identification because scm null mutants show only marginal inflorescence defect, although a stem length abnormality was reported in some scm mutant alleles (Chevalier et al., 2005). This implies that redundant receptors may be acting in this pathway in the absence of SCM function.

Figure 5. (Continued.) SCM::mKD2GFP. E, scm-2/scm-2 SCM::mKD3GFP/SCM::mKD3GFP. F, scm-2/scm-2 SCM::dKD2GFP/+. G, scm-2/scm-2 SCM::mKD1GFP/+ SCM::dKD2GFP/+. H, SCM/scm-2 SCM::dKD2GFP/+. I, SCM/scm-2 SCM::dKD2GFP/SCM::dKD2GFP. J, SCM/SCM SCM::dKD2GFP/+. K and L, SCM/SCM SCM::dKD2GFP/SCM::dKD2GFP. M, SCM/SCM SCM::dKD2/SCM::dKD2. N, scm-2/scm-2 SCM::dKD2/+. O, Flower from SCM/SCM SCM::dKD2GFP/SCM::dKD2GFP with sepal and petal removed to show internal organs. P, Expression of the SCM::GUS reporter gene in the wild-type flower, with an arrow indicating replum expression. Q, SCM/SCM SCM::dKD1/SCM::dKD1. R, scm-2/scm-2 SCM::dKD1/+. S, Quantification of inflorescence size phenotype. The length of inflorescence stems of 8 to 12 mature plants of each line was measured. Error bars represent the SD. Asterisks indicate significant difference from the Columbia wild-type control (WT; P , 0.05, Student’s t test). Bar = 10 mm in A to N, Q, and R; and 1 mm in O and P. Plant Physiol. Vol. 166, 2014


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(Chevalier et al., 2005; Winter et al., 2007; Yadav et al., 2008) and we confirmed that all of the mutated SCM proteins were expressed in the inflorescence meristem in this study. It is notable that the inflorescence defect of dKD2GFP appears to be a qualitative, not a quantitative, effect. Among the lines with varying doses of wild-type SCM and dKD2 SCM kinase-mutant genes, we only observed two phenotypes: a normal-sized inflorescence or a severely reduced inflorescence (Fig. 5). The lack of intermediate phenotypes may be explained if the SCM-associated signaling acts as a trigger to activate (or not activate) an inflorescence regulatory cascade. Our discovery of two additional SCM-related functions emphasizes the wide array of developmental processes that are influenced by the SCM LRR-RLK. A general similarity in the role of SCM in these varied processes appears to be to mediate signaling between adjacent tissues or cells. In the root epidermis, SCM is thought to facilitate interactions between the cortex and epidermis (Kwak et al., 2005); in developing floral organs, SCM mediates intercellular signaling in different cell layers (Yadav et al., 2008); and as proposed here, SCM may enable communication between developing tissues to produce a functional dehiscence zone. Given this general role of SCM, it may be that SCM acts in inflorescence formation by coordinating the differentiation and growth of cells/tissues between layers at/near the inflorescence meristem after floral induction.

The Importance of the SCM Kinase Domain

Figure 6. Expression and localization of intact and mutated SCM-GFP fusion proteins in the inflorescence meristem. The inflorescence meristem of 3-week-old plants in the scm-2 mutant background (A–O) or the wild-type background (P–R) was analyzed by confocal microscopy. The plasma membrane localization of intact SCMGFP (A–C), mKD1GFP (D–F), mKD2GFP (G–I), mKD3GFP (J–L), and dKD2GFP (M–R) was detected. The green fluorescence in A, D, G, J, M, and P indicates SCM-GFP fusion proteins and red in the middle column of B, E, J, K, N, and Q represents FM4-64 fluorescence. Bar = 20 mm.

It is conceivable that the inflorescence defect represents a spurious effect of the mutant SCM kinase constructs and that SCM does not normally participate in inflorescence development. We consider this to be unlikely because many different SCM kinase-mutant proteins (mKD2GFP, mKD3GFP, dKD2GFP, dKD2, and dKD1) generated the same effect. Furthermore, these were driven by the native SCM promoter, rather than an independent strong promoter (e.g. Cauliflower mosaic virus 35S) that would be more likely to generate irrelevant phenotypes. Finally, strong expression of SCM in the inflorescence meristem was independently identified 984

The kinase domain of SCM includes the 12 conserved subdomains and most of the amino acid residues critical for catalytic activity in canonical kinases (Hanks et al., 1988; Fig. 1B). Nevertheless, SCM proteins lack in vitro kinase activity and do not require some of the catalytically critical residues to function (Chevalier et al., 2005; Vaddepalli et al., 2011; this study). Thus, SCM is considered to be one of a growing number of atypical kinases in plants, which possess a nearly complete conserved kinase domain without apparent kinase activity (Castells and Casacuberta, 2007; Gish and Clark, 2011). The reason for the presence of conserved kinase domains in atypical kinases is currently unclear. Here we show that a likely explanation for the conserved kinase domain in SCM is that certain residues are necessary for SCM function in particular pathways. For example, the E686K (mKD2) substitution causes inflorescence defects, but does not affect SCM’s role in root epidermal patterning or fruit development. Our combined analyses suggest that although any particular conserved residue(s) may not be required for SCM function in a single process, each of the conserved residues is likely to be important for SCM to function in at least one of its many signaling processes. This may provide a general explanation for the maintenance of conserved residues in the kinase domain of atypical kinases. Plant Physiol. Vol. 166, 2014



Figure 7. The dKD2GFP protein does not inhibit root epidermal patterning or fruit development. A and B, Root-hair formation in 4-d-old SCM::dKD2GFP seedlings (B) is indistinguishable from the wild type (A). C and D, The expression pattern of the GL2:: GUS transgene in epidermal cells of meristematic zone of 4-d-old wild-type roots (C) is similar to SCM::dKD2GFP roots (D). E, The mature siliques of SCM::dKD2GFP plants exhibit normal dehiscence. Bar = 200 mm in A and B; 50 mm in C and D; and 2 mm in E.

The apparent lack of kinase activity in the SCM protein implies that the intracellular kinase domain mediates signaling by interacting with downstream signaling effectors (Chevalier et al., 2005). In support of this view, other LRR-RLKs in plants are known to facilitate signaling without kinase activity. PANGLOSS1, an atypical LRR-RLK in maize (Zea mays), was found to physically associate with RHO OF PLANTS GTPase in maize extracts (Humphries et al., 2011). FEI1, a kinaseactive Arabidopsis LRR-RLK, does not require kinase activity to rescue the fei1 mutation, and it directly interacts with 1-aminocyclopropane-1-carboxylic acid synthase (Xu et al., 2008). XopN, a virulence factor of Xanthomonas campestris that is translocated into plant cells, is able to bind to the cytosolic domain of TOMATO ATYPICAL RECEPTOR-LIKE KINASE1, an atypical LRR-RLK (Kim et al., 2009).

In addition to the differential effects of certain kinase domain mutants on SCM-associated pathways, we found that other kinase domain mutant proteins caused similar effects in all three developmental processes. For example, the mKD3GFP (G681R) protein did not promote/permit root epidermis patterning, fruit dehiscence, or normal inflorescence formation. At the

Three Distinct SCM Signaling Mechanisms

Our analysis of SCM kinase domain mutants enables us to distinguish distinct signaling requirements for SCM to participate in the three different developmental processes investigated here. Among the four mutants analyzed, we found that root epidermal patterning is sensitive to the mKD3 substitution and the dKD2 kinase domain deletion, fruit dehiscence is sensitive to the mKD3 substitution only, and inflorescence development is sensitive to the mKD2 substitution, the mKD3 substitution, and the dKD2 kinase domain deletion. The differential sensitivity of these SCM-associated processes to the kinase domain alterations implies that SCM signaling is not identical in all SCM-mediated processes; rather, it is likely that distinct signaling mechanisms are used to mediate these three developmental pathways.

Figure 8. The distinct effects of the kinase-altered SCM proteins define three distinct pathways for SCM action during Arabidopsis development. Our work suggests that SCM interacts with three independent pathway-specific factors: Factor A controls root epidermal patterning, factor B regulates fruit dehiscence, and factor C controls inflorescence growth. Arrowheads represent positive interaction/effect, and blunt ends mean negative interaction/effect on the factors.



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other extreme, the mKD1GFP (K525E) protein behaved like the wild-type SCM and had no negative effect on the three processes. These observations likely reflect common requirements of the SCM protein to participate in these three developmental pathways. Given the many SCM-associated developmental processes, SCM may be acting as a general signaling factor that mediates communication between many different cell/tissue layers by interacting with and/or activating cell/tissue-specific intracellular factors. For the processes studied here, these cell/tissue-specific factors would be expected to include a root epidermis-specific factor, a carpel-specific factor, and an inflorescence-specific factor (labeled as A–C in Fig. 8). These proposed pathwayspecific factors may explain the differential sensitivity to kinase domain mutants that we observed in this study. That is, the mKD2GFP mutant protein would be expected to successfully interact with/activate the factor A (in the root epidermis) and the factor B (in the developing carpel/silique), but it would be expected to fail to interact with/activate the factor C (in the developing inflorescence) and instead interfere with this signaling process (Fig. 8). In addition to SCM, other multifunctional LRR-RLKs have been identified in Arabidopsis. For example, the BRI1-ASSOCIATED RECEPTOR KINASE1 functions as a coreceptor for brassinosteroid (with BRI1) and a coreceptor for bacterial flagellin (with FLS2; Li et al., 2002; Nam and Li, 2002; Chinchilla et al., 2007; Heese et al., 2007; Kim et al., 2013). In a similar way, SCM might also perceive multiple ligands using different coreceptors to activate distinct signaling cascades in different parts of the plant.

59-ctc gag att att tgt gta ttg ctg aag-39. To generate the SCM::dKD1 construct, the SCM protein-coding sequence of the SCM::genomic SCM construct was replaced by the dKD1 fragment that lacks the entire kinase domain-coding sequence and most of the juxtamembrane coding sequence (dKD1 retains 17 amino acids of the juxtamembrane domain). The dKD1 fragment was generated by PCR with primers 59-ggt acc atg agc ttt aca aga tgg gaa-39 and 59gga tcc tta acg agc tcc act gta ata tcg-39. All gene constructs described above contain introns and the 662-bp 39 genomic sequence downstream of the stop codon of the SCM coding region. These mutant versions of the SCM::SCMGFP construct (mKD1GFP, mKD2GFP, mKD3GFP, and dKD2GFP) and SCM:: dKD1 were introduced into scm-2 mutant plants harboring the GL2::GUS reporter gene using the Agrobacterium tumefaciens GV3101 strain via the floral dip transformation method (Clough and Bent, 1998). For each experiment, at least three independent transgenic plants were analyzed, and the results of representative lines are shown.

In Vitro Phosphorylation Assay The wild-type SCM kinase domain, the SCM kinase domain with the K525E substitution, and the AtSERK1 kinase domain were subcloned into the pET28a vector. The recombinant proteins were expressed in Escherichia coli and purified by an a His-trapping column (GE Healthcare Life Sciences). Protein extracts were incubated in the reaction buffer (50 mM HEPES, pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 1 mM dithiothreitol, 50 mM ATP, 50 mM NaCl, 0.01% [v/v] Triton X-100, and 25 mCi g-32P-ATP) for 30 min at 30°C. After incubation, Nonidet P-40 and TCA were added, and proteins were collected by centrifugation. The protein pellet was washed and separated in an SDS-PAGE gel. The gel was stained with Coomassie Brilliant Blue and autoradiography was performed.

RT-PCR Total RNA was extracted from 6-d-old shoots of each transgenic plant (QIAGEN), and treated with DNase I (Thermo Scientific) for 30 min at 37°C. The first-strand complementary DNA (cDNA) was synthesized using 1 mg of total RNA and the Maxima first-strand cDNA synthesis kit (Thermo Scientific). PCR was performed with GoTaq (Promega) and each cDNA preparation. For detection of SCM-GFP fusion transcripts, the following mGFP5 primers were used: 59-gga tcc atg agt aaa gga gaa gaa ctt-39 and 59-act agt tta ttt gta tag ttc atc cat gcc-39. For control PCR, the following primers specific for the ELONGATION FACTOR1a gene (At5g60390) were used: 59-cag ggt tga cca ctg agg tt-39 and 59-cat cat ttg gca ccc ttc tt-39.

MATERIALS AND METHODS Arabidopsis Strains and Growth Conditions The Arabidopsis (Arabidopsis thaliana) seeds were sterilized and germinated vertically on agarose-solidified medium as previously described (Schiefelbein and Somerville, 1990). The scm-2 allele (ecotype Columbia of Arabidopsis), with an intragenic transfer DNA insertion (SALK_086357) in the third intron, lacks detectable SCM expression (Kwak et al., 2005). The wild-type GL2::GUS reporter line has been generated using the ecotype Wassilewskija of Arabidopsis (Masucci et al., 1996). Plants were grown in growth chambers at 22°C with a 12-h-light/12-h-dark cycle. For the flowering time analysis, an 18-h-light/6-hdark cycle was used.

Mutagenesis of SCM and Plant Transformation Various point mutations were introduced into the SCM genomic DNA sequence using the Stratagene QuickChange site-directed mutagenesis kit. The SCM genomic DNA fragment corresponding to the kinase domain in the previously described SCM::SCM-GFP translational fusion (Kwak and Schiefelbein, 2008) was excised and used for site-directed mutagenesis. After the point mutation was introduced, the mutated DNA fragment was ligated back into the SCM::SCM-GFP construct to enable the SCM promoter to express GFP-fused mutant proteins. The primers 59-cat gga aag ttt ctt gcg gtg gag aag ctg agc aat acc atc-39, 59-agc ctt ggg gtt gta atg tta aaa ctg ctc act gga cgc aga-39, and 59-cag agc gac gta ttt agc ctt agg gtt gta atg tta gaa ctg-39 were used as the sense primer for mKD1 (K525E), mKD2 (E686K), and mKD3 (G681R) mutagenesis, respectively. To generate the SCM::dKD2GFP construct, the SCM protein-coding sequence of SCM::SCM-GFP was replaced by the dKD2 fragment that lacks the kinase domain-coding sequence. The dKD2 fragment was generated by PCR with primers 59-ggt acc atg agc ttt aca aga tgg gaa-39 and 986

Microscopy, GUS Histochemical Analysis, and Cell Type Analysis Four-day-old roots were analyzed for GFP expression using an LSM510 confocal microscope (Zeiss) as previously described (Kwak and Schiefelbein, 2008). Roots were stained with 10 mg mL21 propidium iodide for 2 min at room temperature. The cells in the inflorescence meristem of 3-week-old plants were stained with FM4-64. Nonhair cell fate was determined by assessing GL2:: GUS reporter expression (Masucci et al., 1996) in 4-d-old roots incubated in GUS staining solution at 37°C for 30 min. GL2::GUS-expressing cells and GL2::GUSnonexpressing cells in the H position and N position of the meristem region of the root tip were counted. Flowers were incubated in GUS staining solution at 37°C for 12 h for SCM::GUS staining.

Protein Structure Analysis The three-dimensional protein structure prediction was conducted on the SCM kinase sequence using the Phyre2 server (Kelley and Sternberg, 2009). Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL data libraries under accession numbers GL2 (At1g79840), SCM (At1g11130), and SERK1 (At1g71830).

ACKNOWLEDGMENTS We thank the Arabidopsis Biological Resource Center for the SALK_086357 insertion line and Christa Barron and Xiaohua Zheng for helpful assistance. Received July 22, 2014; accepted August 15, 2014; published August 18, 2014. Plant Physiol. Vol. 166, 2014



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Profiling dose-dependent activation of p53-mediated signaling pathways by chemicals with distinct mechanisms of DNA damage.

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Galanin receptor 2 utilizes distinct signaling pathways to suppress cell proliferation and induce apoptosis in HNSCC.

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Metformin targets multiple signaling pathways in cancer.

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Compartmentalization of distinct cAMP signaling pathways in mammalian sperm.

Toll-like receptor signaling pathways.

The cold response of CBF genes in barley is regulated by distinct signaling mechanisms.

Cysteine and cysteine-related signaling pathways in Arabidopsis thaliana.

Multiple signaling pathways of orexin receptors.

Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways.

CD3 and CD28 use distinct intracellular signaling pathways.

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Correction: Misregulation of AUXIN RESPONSE FACTOR 8 Underlies the Developmental Abnormalities Caused by Three Distinct Viral Silencing Suppressors in Arabidopsis.

Distinct single-cell signaling characteristics are conferred by the MyD88 and TRIF pathways during TLR4 activation.

Identification of common oncogenic and early developmental pathways in the ovarian carcinomas controlling by distinct prognostically significant microRNA subsets.

Neuropeptidomics Mass Spectrometry Reveals Signaling Networks Generated by Distinct Protease Pathways in Human Systems.