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Mitogen-activated protein kinase regulated by the CLAVATA receptors contributes to shoot apical meristem homeostasis - PubMed

Mitogen-activated protein kinase regulated by the CLAVATA receptors contributes to shoot apical meristem homeostasis

Shigeyuki Betsuyaku et al. Plant Cell Physiol. 2011 Jan.

Abstract

In Arabidopsis, the CLAVATA (CLV) pathway operates in the regulation of the size of the stem cell population in the shoot apical meristem (SAM). CLV3 functions as a small peptide ligand to negatively regulate the expression of the WUSCHEL (WUS) transcription factor through three major receptor kinase complexes of CLV1, CLV2-SUPPRESSOR OF LLP1-2 (SOL2)/CORYNE (CRN) and recently identified RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2)/TOADSTOOL 2 (TOAD2). Aiming to understand the precise molecular details of CLV3 signaling, we investigated the contribution of phospho-signaling, potentially regulated by these kinase complexes, to the CLV pathway. We detected CLV3-triggered CLV1 phosphorylation, which is also conditioned by the rest of the CLV receptors, presumably by their direct association. Our comprehensive analysis of the activities of the respective CLV receptors on mitogen-activated protein kinases (MAPKs) suggested that the precise balanced regulation of MAPK activity by the CLV receptors is likely to be key for SAM homeostasis.

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Figures

Fig. 1
Fig. 1

A schematic diagram of our experimental system using Nicotiana benthamiana. We transiently expressed the CLAVATA components via Agrobacterium tumefaciens in N. benthamiana leaves. We used CLE44–YFP and YFP–YFP as negative controls of CLV3–YFP. All the constructs were C-terminally epitope tagged and were driven under the control of the CaMV 35S promoter. A mixture of Agrobacterium strains carrying the respective CLV constructs as well as the p19 silencing suppressor were infiltrated into leaves of N. benthamiana. The leaf samples were harvested 3 d after infiltration. All the epitope-tagged CLV components used in this study are schematically drawn in a dashed box. The picture shows three stable CLV receptor complexes proposed by a number of studies.

Fig. 2
Fig. 2

CLV1 associates with CLV2, SOL2/CRN and RPK2/TOAD2 in the presence of SOL2/CRN. CLV1-3HS, CLV2-3FLAG, SOL2/CRN-10Myc and RPK2/TOAD2 were co-expressed in N. benthamiana, and total protein extracts were subjected to immunoprecipitation using anti-HA antibody. The resulting immunocomplexes were analyzed by Western blot with anti-HA, anti-Myc or anti-FLAG antibody. CLV1-3HS was immunoprecipitated with CLV2, SOL2/CRN-Myc and RPK2/TOAD2 only when SOL2/CRN-10Myc was also expressed. This experiment was repeated at least twice with a similar result.

Fig. 3
Fig. 3

SOL2/CRN kinase activity is required for SOL2/CRN function in CLV signaling. The stable transgenic sol2 plants expressing either SOL2/CRN-10Myc or SOL2/CRNK146E-10Myc under the control of the CaMV 35S promoter were generated and the resulting plants were analyzed. (A) Immunoblot analysis of 7-day-old seedlings of the respective genotypes. The T2 segregating transgenic populations were analyzed with the wild-type and the sol2 mutant plants. Total protein extracts were detected with anti-Myc. Equal loadings were confirmed by Coomassie Brilliant Blue (CBB) staining of the blot. (B) Semi-quantitative reverse transcription–PCR (RT–PCR) analysis using specific primer sets detecting only SOL2-10Myc fusion transcripts. Total mRNAs were extracted from the same set of plants as in A. The numbers of PCR cycles are shown on the left side of every gel picture. (C) The silique phenotypes of T1 transgenic plants. Five representative siliques from the transgenic plants are shown. Lines 10-2, KD-2 and KD-3 showed the clv-like carpel phenotype of the sol2 mutants, while line 10-1 restored the sol2 defect. The sol2 carpel phenotype was clearly detected by the shape of the siliques.

Fig. 4
Fig. 4

Co-expression of the CLV components detected CLV3-induced mobility shifts of CLV1-3HS and CLV2-3FLAG. CLV1-3HS was expressed with different combinations of CLV receptors in N. benthamiana. Expression levels of the respective receptors and effects of CLV3–YFP co-expression on the CLV receptors were analyzed using immunoblotting. YFP–YFP was used as a negative control of CLV3–YFP co-expression. (A) Immunoblot (8% gel) detected with anti-HA for CLV1-3HS. The CLV3-triggered electrophoretic mobility shift of CLV1 was detected with all combinations of the CLV receptors tested here. (B) Immunoblot (8% gel) with anti-FLAG. CLV2 mobility on SDS–PAGE was also affected by CLV3 co-expression. A clear band shift by CLV3 co-expression is indicated by the blue double-sided arrow. (C) Immunoblot (8% gel) with anti-Myc to detect RPK2/TOAD2-10Myc. (D) Immunoblot (8% gel) detected with anti-Myc for SOL2/CRN-10Myc. (E) Blot (15% gel) with anti-GFP for YFP–YFP expression. (F) Immunoblot (15% gel) with anti-GFP for CLV3–YFP expression.

Fig. 5
Fig. 5

CLV3 specifically triggers CLV1 phosphorylation. (A) Immunoblot analysis of CLV1-3HS protein in the presence/absence of different CLE proteins. The protein extracts from N. benthamiana expressing the constructs (+) were separated by SDS–PAGE (6%) and were detected using anti-HA. (B) Phosphatase treatment of immunoprecipitated CLV1-3HS protein co-expressed with CLV3–YFP. Immunoprecipitated CLV1-3HS protein was mock treated, or treated with recombinant calf intestinal alkaline phosphatase (CIP) or CIP together with phosphatase inhibitors (PIs). The resulting protein samples were separated by SDS–PAGE (6%) and analyzed by immunoblotting with anti-HA. (C) Immunoblot (6% gel) of CLV1-3HS detected with anti-HA. The protein samples from N. benthamian expressing the various constructs shown above were analysed by immunoblotting for CLV1-3HS mobility. A red line is shown to visualize clearly the differences in the CLV1-3HS electrophoretic mobility.

Fig. 6
Fig. 6

CLV3 activates MPK6 and the CLV receptors modulate MPK6 differently. (A) The respective CLV receptors were expressed with or without CLV3–YFP and analyzed by immunoblot and by in-gel kinase assay. All the CLV receptors were also co-expressed together with or without CLV3–YFP and analyzed accordingly. (B) Seven-day-old seedlings of Arabidopsis were mock treated (−) or treated with the MCLV3 peptides (+) for 30 min. The resulting total proteins were analyzed by in-gel kinase assay, IP-kinase assay using anti-MPK6 antibody and immunoblot with anti-MPK6. Relative MPK6 activity is shown.

Fig. 7
Fig. 7

MKK4-KN rescues the clv1-11 defect. Right panels show the whole clv1-11 T2 transgenic plant with DEX-inducible MKK4-KN. Left insets show the carpels treated with or without DEX. Asterisks indicate the rescued siliques with two carpels, whereas older and younger siliques form abnormal clv-like carpels. White bars = 5 mm.

Fig. 8
Fig. 8

A schematic model of the CLV receptor activities in MAPK regulation and the expression domains of the CLV components. (A) The activities of the CLV receptors on MAPK regulation and their association specificities detected in this study are schematically shown as discussed in the text. Important CLV signaling components, which might be involved in MAPK regulation, are also shown at the position where they possibly play roles (KAPP, ROP and POL shown in a purple circle). (B) The expression domains of the CLV components in the SAM are schematically shown (Clark et al. 1997, Mayer et al. 1998, Fletcher et al. 1999, Jeong et al. 1999, Kinoshita et al. 2010). The expression domains of CLV3, WUS, CLV1, CLV2-SOL2/CRN and RPK2/TOAD2 are indicated as a merged picture on the top and are also shown individually at the bottom.

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