AtPP2C52 (GenBank Accession No.: NP_680572) was identified as an AGB1-interacting protein (Tsugama et al., 2012a). Coimmunoprecipitation (Co-IP) was used to confirm the interaction of AtPP2C52 and AGB1 in vitro (Figure 1). Myc-tagged AGB1 (Myc:AGB1), HA-tagged AtPP2C52 (HA: AtPP2C52) and HA epitope tag (HA) were synthesized in vitro in a rabbit reticulocyte lysate system. Either HA epitope tag or HA:AtPP2C52 was mixed with Myc:AGB1, and then precipitated by anti-HA antibody. Subsequently, G Sepharose was added. After incubation, Myc:AGB1 in the elutant from the G Sepharose was analyzed by immunoblotting using anti-Myc antibody. Specific signals of Myc:AGB1 were detected only when AtPP2C52 was present (Figure 1), indicating that AtPP2C52 interacts with AGB1 in vitro.

Figure 1 AtPP2C52 interacts with AGB1 in vitro

To analyze the temporal-spatial expression pattern of AtPP2C52, transgenic plants expressing a promoter-reporter fusion gene (P_AtPP2C52::GUS) were used. P_AtPP2C22::GUS was expressed in almost all the plant organs (Figure 2). In 4-day-old seedlings, P_AtPP2C52::GUS was predominantly expressed in vascular, root tip and apical meristem (Figure 2A; Figure 2B). In 3-week-old plants, P_AtPP2C52::GUS was evident in the whole plant (Figure 2C). The expression of P_AtPP2C52::GUS was still higher in vascular and apical meristem in this stage. In adult plants, P_AtPP2C52::GUS was found in all the organs of flower, excepting the anther (Figure 2E; Figure 2F). The expression level of P_AtPP2C52::GUS was lower in the sporangia (Figure 2G; Figure 2H).

 

Three site-directed mutants of AtPP2C52 (AtPP2C52^G99D, AtPP2C52^G105D and AtPP2C52^{DGH102-104ERN}) were generated (Figure 3A). These mutated sites were highly conserved, and they are involved in the PP2C active site (Das et al., 1996; Sheen, 1998). The mutated sequences encoded unrelated amino acids. None of these mutations affected the molecular weight of these mutant proteins (Figure 3B). However, all of these mutations abolished the interaction between AGB1 and AtPP2C52 (Figure 3C).


1.4 Potential substrates of AtPP2C52

To further identify compartments of the signaling pathway mediated by AtPP2C52, full-length AtPP2C52 was used as the bait in Y2H screening. Even on high-stringency selection media, more than 2500 positive clones were obtained and 300 clones were sequenced. Among them, a proteasome maturation factor, UMP1, and a cysteine proteinase, RD21a, were used for further analysis.

The interaction of AtPP2C52 with either UMP1 or RD21a was confirmed by Y2H (Figure 4). AtPP2C52^G99D and AtPP2C52^{DGH102-104ERN} mutants failed to interact with UMP1 in Y2H (Figure 4A). G105D mutation did not affect the Y2H interaction between AtPP2C52 and UMP1 (Figure 4A). All of these mutations abolished the Y2H interaction between AtPP2C52 and RD21a (Figure 4B). The interactions were examined by a BiFC assay in Arabidopsis protoplast. 

The ORFs of AGB1, UMP1 and RD21a were fused downstream of the nYFP and the ORF of AtPP2C52 was fused upstream of the cYFP. BiFC signals of nYFP-fused AGB1 and cYFP-fused AtPP2C52 were detected in the peripheral region of Arabidopsis mesophyll protoplasts (Figure 5) as previously described (Tsugama et al., 2012a). BiFC signals of nYFP-fused UMP1 and cYFP-fused AtPP2C52 were also detected in the peripheral region (Figure 5), suggesting that AtPP2C52 interacted with UMP1 in the plasma membrane. AtPP2C52 interacted with RD21a not only in the plasma membrane but also in the nucleus (Figure 5). These results suggest that RD21a and UMP1 are the potential substrates of AtPP2C52.

Figure 5 AtPP2C52 interacted with UMP1and RD21a in BiFC assays in Arabidopsis protoplasts

Here we proved that P_AtPP2C52::GUS was evident in the whole plant in 3-week-old plants (Figure 2C), and P_AtPP2C52::GUS was found in most of the flower organs (Figure 2D~F), indicating that AtPP2C52 gene has a broad expression pattern as other PP2C genes in Arabidopsis (Xue et al., 2008). On the other hand, the expression of P_AtPP2C52::GUS was predominantly found in vascular, root tip and apical meristem in 4-day-old seedlings (Figure 2A; Figure 2B). The expression of P_AtPP2C52::GUS was lower in the anther (Figure 2D~Figure 2F) and sporangia (Figure 2G; Figure 2H). These results suggest that the expression of AtPP2C52 gene was in a tissue specific manner at some level.

2.2 Mutational analysis of AtPP2C52

These mutated sites of AtPP2C52 were highly conserved in PP2C family. ABI1 is a well-known member belonging to PP2C Group A (Sheen, 1998). Three AtPP2C52 mutants, G99D, DGH102-104ERN and G105D, corresponded to the ABI1 mutants, G174D, DGH177-179KLN and G180D, respectively (Sheen, 1998). ABI1 mutations (G174D and DGH177-179KLN) abolished the ability of ABI1 to block ABA-inducible transcription. G174D and DGH177-179KLN were identified as true null mutations of ABI1 owing to dramatic changes in amino acids charges near to or at the PP2C active site. G180D mutation abolished PP2C activity of ABI1. G180D mutant was as effective as wild-type ABI1 in abolishing the ABA-inducible transcription, suggesting G180D mutant blocks ABA responses through a dominant interfering effect without PP2C activity. Here, G99D and DGH102-104ERN mutations of AtPP2C52 abolished the ability of AtPP2C52 to interact with AGB1, UMP1 and RA21a in Y2H assay (Figure 3C, Figure 4). G105D mutation did not affect the Y2H interaction between AtPP2C52 and UMP1 (Figure 4A). These results suggest that these residues may essential for AtPP2C52 to bind specific targets, such as UMP1, RA21a and AGB1. G105D may play a different role from G99D and DGH102-104ERN.

In conclusion, physical interactions between AtPP2C52 with UMP1, RA21a and AGB1 were confirmed. Mutational analysis shown that G99D, DGH102-104ERN and G105D are essential for AtPP2C52 to bind specific targets, such as UMP1, RA21a and AGB1.

3 Materials And Methods

Arabidopsis Col-0 plants were used. Seeds were surface sterilized in 70% (v/v) ethanol solution for 30s, then sterilized in 0.25% (v/v) sodium hypochlorite containing 1% (v/v) Tween X-100 and washed four times in sterile distilled water. Afterward, seeds were sowed on MS plates containing half MS basal salts (Wako) and 1% (w/v) sucrose, pH5.7, solidified with 0.8% (w/v) agar (Wako). Plates were sealed and incubated in 4℃ for 72h in darkness, then transferred to growth chamber incubated at 22℃. Light intensity is 120 μmoL•m^-2•s^-1. After ten days of growth, plants were transferred onto rockwool cubes and grown further with 0.2×MS solution regularly supplied.

Y2H experiments were performed as previously described (Tsugama et al., 2012a). cDNA clone RAFL18-04-O14 of AtPP2C52 (AT4G03415) was obtained from RIKEN BRC Experimental Plant Division (Seki et al., 2002). The open reading frame (ORF) of AtPP2C52 was amplified by PCR using the cDNA clone as template and the following primer pair: 5'-CCCGAATTCTCTAGAATGGGGGGTTGTGTGTCGAC-3' (EcoRI and XbaI sites are underlined) and 5'-GGGCTCGAGGAGTCTTCGATTTCTCTTCAGAG-3' (XhoI site is underlined). The PCR products were digested by EcoRI and XhoI, and cloned into the EcoRI/XhoI site of pGADT7-rec, generating pGAD-AtPP2C52.

The point mutations of AtPP2C52 gene were generated by PCR using PrimeSTAR (TaKaRa) with wild type AtPP2C52 cDNA as template. For the point mutation of AtPP2C52^G99D, PCR was performed using the following primer pair: G99D-Fw 5'-GTGACATTTTGTGATGTATTTGATGGTCATGGTCC-3' and AtPP2C52-Rv 5'-GAGTCGGATCCTCAAGTCTTCGATTTCTCTTC-3’ (BamHI site is underlined), generating 3'- terminus of AtPP2C52^G99D. To generate 5’- terminus of AtPP2C52G99D, PCR was performed using the following primer pair: AtPP2C52-Fw 5'-GAGTCGAATTCATGGGGGGTTGTGTGTC-3' (EcoRI site is underlined) and G99D-Rv 5’-GACCATCAAATACACCACAAAATGTCACATCTTCAGAC-3’. Subsequently, the mixture of 3’- and 5’-terminus of AtPP2C52^G99D was used as template for PCR using primer pair AtPP2C52-Fw and AtPP2C52-Rv, generating full-length AtPP2C52^G99D. The PCR products of AtPP2C52^G99D were digested by EcoRI and BamHI, and cloned into the EcoRI/BamHI site of pGADT7-rec, generating pGAD-AtPP2C52^G99D. For the point mutation of AtPP2C52^G99D, PCR was performed using the following primer pair: G105D-Fw 5’-GATGGTCATGATCCTTATGGCCATCTTGTTGCTCG-3’ and AtPP2C52-Rv, generating 3’-terminus of AtPP2C52^G105D.

To generate 5'-terminus of AtPP2C52^G105D, PCR was performed using the following primer pair: AtPP2C52-Fw and G105D-Rv 5’-GCCATAAGGATCATGACCATCAAATACACCACAAAATG-3'. Subsequently, the mixture of 3'- and 5'-terminus of AtPP2C52^G105D was used as template for PCR using primer pair AtPP2C52-Fw and AtPP2C52-Rv, generating full-length AtPP2C52^G105D. The PCR products of AtPP2C52^G105D were digested by EcoRI and BamHI, and cloned into the EcoRI/BamHI  site of pGADT7-rec, generating pGAD-AtPP2C52^G105D.

For the point mutation of AtPP2C52^{DGH102-104ERN}, PCR was performed using the following primer pair: DGH102-104ERN-Fw 5’-GGTGTATTTGAACGTAATGGTCCTTATGGCCATCTTG-3’ and AtPP2C52-Rv, generating 3’-terminus of AtPP2C52^{DGH102-104ERN}. To generate 5’-terminus of AtPP2C52^{DGH102-104ERN}, PCR was performed using the following primer pair: AtPP2C52-Fw and DGH102-104ERN-Rv 5’-CATAAGGACCATTACGTTCAAATACACCACAAAATGGC-3’. Subsequently, the mixture of 3’- and 5’-terminus of AtPP2C52 DGH102-104ERN was used as template for PCR using primer pair AtPP2C52-Fw and AtPP2C52-Rv, generating full-length AtPP2C52^{DGH102-104ERN}. The PCR products of AtPP2C52 DGH102-104ERN were digested by EcoRI and BamHI, and cloned into the EcoRI/BamHI site of pGADT7-rec, generating pGAD-AtPP2C52^{DGH102-104ERN}. These mutations were confirmed by sequencing and then used for yeast transformation.

Full-length cDNA of UMP1 (AT1G67250) and RD21a (AT1G47128) were ordered from ABRC and used as PCR templates for following plasmids construction. The ORF of UMP1 was amplified by PCR using the cDNA clone as template and the following primer pair: 5’-GAGTCGAATTCATGGAGTCTGAGAAAAAGATAGCTCATG-3’ (EcoRI site is underlined) and 5’-GAGTCGGATCCTTACATGAAACTTGGGTAAATCGG-3’ (BamHI site is underlined). The PCR products were digested by EcoRI and BamHI, and cloned into the EcoRI/BamHI site of pGADT7-rec, generating pGAD-UMP1. The ORF of RD21a was amplified by PCR using the cDNA clone as template and the following primer pair: 5’-GAGTCGAATTCATGGGGTTCCTTAAGCCAACCATGGC-3’ (EcoRI site is underlined) and 5’-GAGTCCCTAGGTTAGGCAATGTTCTTTCTGCCTTGTGACCAG-3’ (BamHI site is underlined). The PCR products were digested by EcoRI and BamHI, and cloned into the EcoRI/BamHI site of pGADT7-rec, generating pGAD-RD21a. 

Yeast transformation using yeast strain AH109 and screening were performed by Matchmaker Gold Yeast Two-Hybrid System (Clontech). At least 5 colonies grown on the SD media lacking leucine and tryptophan (SD/-Leu/-Trp), were streaked on the SD/-Leu/-Trp and the SD media lacking leucine, tryptophan, adenine, and histidine (SD/-Trp/-Leu/-His/-Ade). Photos were taken after the yeasts were cultured for 3-5 days. The experiments were performed for three times. 

3.3 In vitro Coimmunoprecipitation (Co-IP) 

Proteins were expressed by TNT Quick Coupled Transcription/Translation Systems (Promega). Plasmid DNA pGBK-AGB1, pGAD-AtPP2C52, pGAD-AtPP2C52^G99D, pGAD-AtPP2C52^G105D, pGAD-AtPP2C52^{DGH102-104ERN} and pGADT7-rec vector were used to synthesize proteins Myc-tagged AGB1, HA-tagged AtPP2C52 and HA epitope tag, respectively. Co-IP was carried out using anti-HA antibody (MBL), anti-Myc antibody (MBL) and protein G Sepharose (GE Healthcare) following the MATCHMAKER Co-IP Kit User Manual (Cat No. 630449, 2003).

3.4 Bimolecular Fluorescence Complementation (BiFC) assay

The vectors for BiFC assay were constructed by replacing GFP in the vector pBS-35SMCS-GFP (Tsugama et al., 2012b) with the N-terminus (154 amino acids) or the C-terminus (80 amino acids) of YFP, generating pBS-35SMCS-nYFP and pBS-35SMCS-cYFP, respectively. The pGAD-AtPP2C52 was digested by XbaI and XhoI, and the resultant ORF fragments of AtPP2C52 were inserted into the XbaI/SalI site of pBS-35SMCS-cYFP, generating pBS-35S-AtPP2C52-cYFP. The ORF of UMP1 without stop codon was amplified by PCR using the cDNA clone as template and the following primer pair: 5'- GAGTCGGTACCATGGGGTTCCTTAAGCCAAC-3' (KpnI site is underlined) and 5'-CTCGAACTAGTGGCAATGTTCTTTCTGC-3' (SpeI site is underlined). The PCR products were digested by KpnI and SpeI, and cloned into the KpnI/SpeI site of pBS-35SMCS-nYFP, generating pBS-35S-nYFP-UMP1. The ORF of RD21a without stop codon was amplified by PCR using the cDNA clone as template and the following primer pair: 5'-GAGTCGGTACCATGGAGTCTGAGAAAAAGATAGC-3' (KpnI site is underlined) and 5'-CTCGAACTAGTCATGAAACTTGGGTAAATCGG-3' (SpeI site is underlined). The PCR products were digested by KpnI and SpeI, and cloned into the KpnI/SpeI site of pBS-35SMCS-nYFP, generating pBS-35S-nYFP-RD21a. Arabidopsis protoplast isolation and transformation were conducted as described (Wu et al., 2009). Plasmids DNA were introduced into onion epidermal cells by a bombardment system (Bio Red, PDS-1000). Images were processed using Canvas X software (ACD Systems) and enhanced using Photoshop CS4 software (Adobe). 

3.5 Preparation of chimeric constructs

An approximately 2 kb upstream promoter sequence of AtPP2C52 (P_AtPP2C52) was cloned from an Arabidopsis genetic DNA by using primer pair: 5’-GGATCCCGGGATGAATCATGTAGGTGAC-3’ (SmaI site is underlined) and 5’-CCCTCTAGATGTTTAATCCCAGCCTAGA-3’ (XbaI site is underlined). The PCR products were double-digested with SmaI/XbaI and used to replace the CaMV 35S promoter of pBI121 vector (Clontech, Bevan, 1984), generating pBI121-P_AtPP2C52::GUS. 

3.6 Plant transformation

Arabidopsis Col-0 plants were transformed by Agrobacterium tumefaciens-mediated transformation using the floral-dip method (Clough and Bent, 1998). The Agrobacterium strain used was EHA105 harboring binary vector pBI121 carrying the P_AtPP2C52::GUS fusion gene.

3.7 GUS histochemical analysis

For GUS histochemical characterization of the P_AtPP2C52::GUS lines, several developmental stages were examined. Samples were treated with 90% acetone for 30 min at 4℃, immersed in a staining solution (5 mmol/L X-gluc, 0.1% Triton X-100, 0.5 mmol/L K3Fe(CN)6, 0.5 mmol/L K₄Fe(CN)₆, 10 mmol/L Na₂EDTA and 50 mmol/L sodium phosphate buffer, pH 7.0), vacuum infiltrated for more than 1 hour, then incubated at 37℃. After staining, samples were cleared by several changes of 70% ethanol. Photographs were enhanced using Photoshop CS4 software (Adobe).

Author Contributions

TetsuoTakano and Shenkui Liu are responsible for overall planning and management. Collecting data and article writing were completed by Hua Liu and Daisuke Tsugama. Manuscript was completed by TetsuoTakano and Hua Liu. 

Acknowledgements

This work is supported by China Scholarship Council (CSC).

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