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First published online March 27, 2009; 10.1104/pp.109.135335 Plant Physiology 150:144-156 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Identification and Functional Characterization of Inhibitor-3, a Regulatory Subunit of Protein Phosphatase 1 in Plants1,[W],[OA]Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Fukuoka 812–8581, Japan
Protein phosphatase 1 (PP1) is a eukaryotic serine/threonine protein phosphatase, and mediates diverse cellular processes in animal systems via the association of a catalytic subunit (PP1c) with multiple regulatory subunits that determine the catalytic activity, the subcellular localization, and the substrate specificity. However, no regulatory subunit of PP1 has been identified in plants so far. In this study, we identified inhibitor-3 (Inh3) as a regulatory subunit of PP1 and characterized a functional role of Inh3 in Vicia faba and Arabidopsis (Arabidopsis thaliana). We found Inh3 as one of the proteins interacting with PP1c using a yeast two-hybrid system. Biochemical analyses demonstrated that Arabidopsis Inh3 (AtInh3) bound to PP1c via the RVxF motif of AtInh3, a consensus PP1c-binding sequence both in vitro and in vivo. AtInh3 inhibited the PP1c phosphatase activity in the nanomolar range in vitro. AtInh3 was localized in both the nucleus and cytoplasm, and it colocalized with Arabidopsis PP1c in these compartments. Disruption mutants of AtINH3 delayed the progression of early embryogenesis, arrested embryo development at the globular stage, and eventually caused embryo lethality. Furthermore, reduction of AtINH3 expression by RNA interference led to a decrease in fertility. Transformation of the lethal mutant of inh3 with wild-type AtINH3 restored the phenotype, whereas that with the AtINH3 gene having a mutation in the RVxF motif did not. These results define Inh3 as a regulatory subunit of PP1 in plants and suggest that Inh3 plays a crucial role in early embryogenesis in Arabidopsis.
Reversible protein phosphorylation, which is regulated by the activities of protein kinase and phosphatase, is an important mechanism of the posttranslational regulation of cellular and developmental processes. Protein phosphatase 1 (PP1) is a major member of the PPP family of Ser/Thr protein phosphatases (PP1, PP2A, PP2B, PP4, PP5, PP6, and PP7) and is ubiquitously distributed in higher eukaryotes (Barton et al., 1994  ; Cohen, 1997, 2002). In animals, PP1 has been shown to regulate diverse cellular processes, such as cell cycle progression, transcription, protein synthesis, carbohydrate metabolism, muscle contraction, and neuronal signaling (Bollen, 2001; Cohen, 2002; Ceulemans and Bollen, 2004).
Recently, we have demonstrated that PP1 functions as a positive regulator in the signaling of blue light-dependent stomatal opening in plants, a process essential for photosynthetic CO2 fixation and transpiration (Takemiya et al., 2006
PP1 consists of a PP1c and multiple distinct regulatory subunits that specify the catalytic activity, substrate specificity, and subcellular localization of the enzyme (Bollen, 2001
In animals, at least 45 genes that encode genuine or putative PP1 regulatory subunits have been identified, and this large set of regulatory subunits forms a variety of distinct holoenzymes with PP1c and enables PP1 to be involved in diverse cellular processes (Bollen, 2001 In this study, we identified inhibitor-3 (Inh3) in V. faba and Arabidopsis as a regulatory subunit for PP1. Inh3 interacted specifically with PP1c, inhibited the PP1c catalytic activity, and was localized in both the nucleus and the cytosol. We also demonstrated that Inh3 plays an essential role in embryo development.
Isolation of a Regulatory Subunit of PP1 by Yeast Two-Hybrid Screening
We carried out a yeast two-hybrid screen using VfPP1c-1, a PP1c expressed in V. faba, as bait (Takemiya et al., 2006
Database searches revealed that this protein is a homolog of human Inh3 and yeast phosphatase inhibitor 1 (Ypi1), which are PP1 regulatory subunits (Zhang et al., 1998 ; García-Gimeno et al., 2003). The homologous proteins were found in Arabidopsis, Medicago truncatula, and O. sativa (Fig. 1A). They contained conserved regions in the middle of the sequences, where we identified two putative RVxF motifs, KVSW in Vicia, Arabidopsis, and Medicago and KVTW in rice. These proteins are conserved in eukaryotes, including fungi, protozoa, insects, nematode, vertebrates, and mammals (Fig. 1B). Therefore, the PP1c-interactive protein of VfPRS1 was renamed VfInh3.
The interaction of VfInh3 with VfPP1c-1 was revealed by the β-galactosidase activity in the yeast two-hybrid system. Strong β-galactosidase activity was observed when VfInh3 was coexpressed with VfPP1c-1, but not with VfPP2Ac-1, a PP2A catalytic subunit from Vicia (Fig. 2A ). Human inhibitor-2, a known PP1 regulatory subunit in mammals, interacted with VfPP1c-1 to the same degree (Fig. 2A). To determine whether the interaction between VfInh3 and PP1c is mediated by KVSW (as RVxF motif), we substituted Ala for the Trp-59 in VfInh3. The resulting mutant protein of VfInh3-W59A lost the ability to bind to VfPP1c-1 (Fig. 2A).
We cloned various catalytic subunits of the PPP family of Ser/Thr protein phosphatases from Arabidopsis and investigated the interaction of these subunits with Arabidopsis Inh3 (AtInh3; At2g31305) using the yeast two-hybrid system (Fig. 2B). AtInh3 bound to all nine PP1c isoforms (Type One Protein Phosphatase [TOPP]) but did not bind to any of the other catalytic subunits (i.e. PP2A, PP4, PP5, PP6, or PP7; Fig. 2B). Substitution of Ala for Trp-43 in the RVxF motif of AtInh3 abolished the binding ability of AtInh3 to PP1cs (Fig. 2B).
The interaction between Inh3 and PP1c was further tested by in vitro pull-down assays using recombinant Inh3 and PP1c (Fig. 2C). That the molecular masses of recombinant VfInh3 (22.3 kD) and AtInh3 (19.0 kD) were higher than the predicted values of 13.8 and 10.5 kD, respectively, was considered to be due to the high hydrophilicity of the two proteins (Zhang et al., 1998 We also observed a binding between Inh3 and PP1c in vivo. Wild-type Columbia-0 (Col-0) plants were transformed with a full-length AtINH3 or AtINH3-W43A fused with a 3xFLAG under a native promoter. The PP1c was coimmunoprecipitated with 3xFLAG:AtInh3 but not with 3xFLAG:AtInh3-W43A by anti-FLAG antibodies (Fig. 2F). Based on these results in vitro and in vivo, we concluded that Inh3 functions as a PP1 regulatory subunit in plant cells.
We investigated the effect of Inh3 on PP1c catalytic activities using a [32P]labeled myelin basic protein as a substrate. Both VfInh3 and AtInh3 strongly inhibited the dephosphorylation activities of PP1c in a concentration-dependent manner, with half-inhibitory concentrations (IC50) of 0.50 and 0.75 nM for VfInh3 and AtInh3, respectively (Fig. 3 ). When the same assay was performed using VfInh3-W59A and AtInh3-W43A, which lost the binding ability to PP1c (Fig. 2), the dephosphorylation activities were not greatly affected by these mutant proteins (Fig. 3). These results suggest that plant Inh3 functions as an activity modulator of PP1c.
Inh3 Was Localized in the Nucleus and the Cytoplasm
We next studied the subcellular localization of Inh3 by transient expression of AtInh3 fused to a green fluorescent protein (sGFP:AtINH3) in Vicia guard cells after particle bombardment as well as the coexpression of the nuclear localization site of cryptochrome 2 (CRY2) fused to a red fluorescent protein (CRY2-
If AtInh3 binds to PP1c in vivo, these two proteins should be colocalized. To examine this, nine PP1c isoforms of Arabidopsis were fused to mCherry (mCherry:TOPP) and transiently coexpressed with AtInh3 or AtInh3-W43A. All nine PP1c isoforms were colocalized with AtInh3 in both the nucleus and cytoplasm (Fig. 4B). In contrast, the efficient nuclear localization of PP1c was diminished when PP1c was coexpressed with AtInh3-W43A, although AtInh3-W43A was localized in both the nucleus and the cytoplasm (Fig. 4C). These results support an in vivo interaction of AtInh3 with PP1c via the RVxF motif.
Expression of Vicia and Arabidopsis INH3 genes was determined by reverse transcription (RT)-PCR in guard cell protoplasts, mesophyll cell protoplasts, leaves, stems, and roots. Transcripts of VfINH3 and AtINH3 were found in all cell/tissue types and particularly in guard cell protoplasts (Fig. 5, A and B ).
We generated transgenic plants expressing the GUS reporter gene driven by the AtINH3 promoter (the 2.2-kb region upstream from the initiator ATG). Blue staining was detected in several organs, including roots and aerial parts (Fig. 5C). In aerial parts, the staining was found in photosynthetic and reproductive organs and was particularly strong in trichomes and the veins of cotyledons and leaves (Fig. 5, D and E). In agreement with the results of the RT-PCR analysis, the staining was found in guard cells (Fig. 5F). The staining was also found in the filaments and stigma of flowers (Fig. 5G), in both the embryo proper (globular stage) and endosperm of developing seeds (Fig. 5H) and in the root tips and steles of root (Fig. 5, I and J). Finally, the staining was visible in young seedlings (Fig. 5K).
Since the expression of AtINH3 was observed preferentially in guard cells, we initially attempted to characterize the physiological function of Inh3 in guard cells using Arabidopsis T-DNA insertion lines. We obtained two T-DNA insertion lines from the SALK and SAIL collections and designated the corresponding alleles inh3-1 (SALK_044593) and inh3-2 (SAIL_806_C02), respectively. The insertions were located in the second exon of AtINH3, 146 and 1 bp downstream of the ATG initiation codon in inh3-1 and inh3-2, respectively (Fig. 6A ). A PCR-based screen using gene-specific primers and the T-DNA left border primer revealed that all the plants from the T3 or T4 generation were the heterozygous or wild-type strains, and there were no homozygous plants (Fig. 6, B and C). All the heterozygous plants were morphologically similar to the wild-type plants and did not show any impairment in stomatal responses (data not shown). Segregation analyses of >100 self-pollinated heterozygous (parent) plants revealed that there was a 2:1 ratio of T-DNA heterozygous plants to wild-type plants (Table I ), suggesting that a homozygous insertion confers the lethality and/or reduced gametophytic transmission. To test whether the insertion was transmitted normally through both male and female gametophytes, we reciprocally crossed inh3-1 with the wild type and screened F1 plants for the T-DNA insertion by PCR. Transmission of the T-DNA insertion occurred through both gametophytes (Table I).
In Arabidopsis, seeds within a single silique develop at a similar rate. If the homozygous insertion in AtINH3 causes embryo lethality, the self-pollinated seeds from heterozygous inh3 mutants would be expected to include aborted seeds. In wild-type siliques, all seeds developed normally and were green in color (Fig. 6D). However, the heterozygous lines of inh3-1 and inh3-2 produced 24.6% and 22.2% white or brown shriveled seeds, respectively (Table II ), and the segregation ratio of normal to abnormal seeds was approximately 3:1, suggesting that these aborted seeds might have been the missing homozygous mutants. Furthermore, the mature dried seeds from wild-type plants exhibited a light brown color and were uniform in shape, whereas the seeds from heterozygous inh3 mutants contained a number of shriveled and dark brown seeds (Supplemental Fig. S3). Taken together, these results suggest that the disruption of AtINH3 causes embryo lethality in Arabidopsis. We thus focused our attention on the role of Inh3 in early embryogenesis.
AtINH3, But Not AtINH3-W43A, Complemented the Embryo-Lethal Phenotype of the inh3 Mutant We complemented the inh3-1 mutant to demonstrate homozygous lethality using a construct containing both the full-length AtINH3 and the hygromycin resistance gene as a marker (Fig. 7A ). We selected T1 plants that contained the heterozygous inh3-1 T-DNA by PCR-based screen (data not shown). The complementation was tested on the hygromycin-resistant T2 plants by PCR using sense (S2) and antisense (AS2) primers, which amplified both endogenous and transgenous AtINH3. The individual PCR products were distinguished by digesting them at the BamHI site that was introduced in the transgene. Among the nine randomly selected hygromycin-resistant T2 plants, three did not possess the endogenous AtINH3 (Fig. 7B, lanes 1, 4, and 7); thus, these three lines were homozygous for the inh3-1 allele. The rest were either heterozygous inh3-1 plants having both endogenous and transgenous AtINH3 (Fig. 7B, lanes 2, 5, 6, and 9) or wild-type plants having the transgene (Fig. 7B, lanes 3 and 8). The siliques of heterozygous inh3-1 plants possessing the transgenous AtINH3 produced almost no white or shriveled brown seeds, and nearly all the seeds exhibited fully developed and uniformly sized embryos (Fig. 7C). Therefore, the embryo-lethal phenotype of the homozygous inh3-1 mutant was fully complemented by the AtINH3 transgene.
The complementation of the inh3-1 embryo lethality by the AtINH3 gene was expected to be due to restoration of the regulation of PP1c via Inh3. It is also possible that Inh3 itself has an essential role in embryogenesis that is independent of PP1c regulation. To test this alternative, we introduced AtINH3-W43A, which lost the ability to bind to PP1c (Fig. 2), into the inh3-1 mutant and inspected the embryo phenotype. We randomly selected nine individual T2 lines and found that there was no homozygous mutant for inh3-1 (data not shown) and that all these heterozygous inh3-1 plants possessing the transgenous AtINH3-W43A produced the aborted seeds (Fig. 7D). The results may support the possibility that the PP1c-Inh3 complex has an important role in early embryogenesis.
We next compared the development of the wild-type plants with that of the mutants during seed maturation. Siliques on 4 to 7 d after flowering (DAF) from wild-type and heterozygous inh3-1 plants were dissected, and the cleared seeds were inspected by light microscopy. The wild-type embryos developed from the globular to the bent cotyledon stage (Table III ). The majority of the embryos were in the early and late heart developmental stages at 4 DAF, the late heart and torpedo stages at 5 DAF, the torpedo stage at 6 DAF, and the bent cotyledon stage at 7 DAF. In heterozygous inh3-1 embryos, approximately 25% of the plants were arrested at the octant-dermatogen and globular stages, whereas the rest of the embryos developed similarly to the wild type (Table III). The seeds of arrested embryos displayed a white color in the early developmental stages due to the depletion of green embryos and later degenerated to show a dark brown color. Figure 8 presents images of defective embryos that appeared in the silique of heterozygous inh3-1 and normal embryos in the wild-type plants. In heterozygous inh3-1, a quarter of embryos grew much slower than the rest and remained in the eight-cell stage at 3 DAF and the globular stage at 4 DAF, whereas the wild-type embryos were in the globular stage at 3 DAF and the early heart stage at 4 DAF. Although embryo development was delayed in the mutant in these stages, embryo patterning appeared not to be largely changed. From 5 to 6 DAF, while the defective embryos continued to grow, they never developed beyond the globular stage and began to show irregular surfaces. At 7 DAF, the defective embryos stopped developing altogether.
Suppression of AtINH3 by RNA Interference Resulted in Reduced Fertility To further elucidate the function of Inh3, we generated transgenic plants expressing AtINH3-RNA interference (RNAi) constructs. A full-length coding region of the AtINH3 was cloned in both a sense and an antisense orientation and was used for transforming Arabidopsis Col-0 plants. We obtained only three transformants, probably due to the embryo lethality in the plants. All these transformants displayed normal vegetative growth and development, but they also exhibited reduced fertility. The siliques of RNAi plants were much shorter than those of wild-type plants and produced a reduced number of seeds (Supplemental Fig. S4A). In accord with the impaired phenotypes, the AtINH3 transcript was detected in immature siliques of wild-type plants, whereas the expression levels were greatly reduced in the AtINH3-RNAi lines (Supplemental Fig. S4B).
Identification of Inh3 as a Regulatory Subunit for PP1
In animals, the function of PP1 is determined by PP1c activity through the control of regulatory subunits, and a large number of regulatory subunits for PP1c have been identified (Bollen, 2001
We investigated the biochemical properties of plant Inh3 and elucidated that Inh3 regulates the catalytic activity of PP1c. Human Inh3 was initially isolated by its physical interaction with PP1c, and biochemical analysis has shown that the recombinant protein is a heat-stable inhibitor protein of PP1c (Zhang et al., 1998
In animals, a number of PP1 regulatory subunits that regulate PP1c activity (i.e. other than Inh3) have been identified. Heat-stable inhibitory activities toward PP1c have also been detected in the extracts of Arabidopsis and Zea mays root (Lin et al., 1998
Studies investigating human Inh3 by immunostaining and the GFP fusion system have shown that Inh3 is localized in the nucleoli and the centrosomes (Huang et al., 2005
Inh3 was initially discovered as an inhibitor of PP1c catalytic activity in humans (Zhang et al., 1998
An important finding in our study was that the lethal phenotype of the inh3 mutant was rescued by wild-type AtINH3, but not by the mutant AtINH3-W43A, which lost its ability to bind and inhibit the PP1c. This result suggests that Inh3 exerts its function by binding to PP1c in embryogenesis, and it is also possible that the PP1c catalytic activity may be regulated by Inh3 via the binding. We tried to measure the PP1c activity in the wild-type and inh3 mutant embryos, but we could not determine the phosphatase activities because of the scarcity of mutant embryo preparations. We also identified the isoforms of Arabidopsis PP1c expressed during early embryogenesis and found that eight of nine PP1c isoforms were expressed in young siliques (Supplemental Fig. S5). This made it difficult to identify the functional role of specific isoforms of PP1c in embryogenesis. Further studies will be needed to determine the regulatory role of Inh3 in embryogenesis.
What, then, is the role of the PP1c-Inh3 complex in early embryogenesis? Both the inh3 mutant and the wild-type showed similar early embryo pattern formation, but then embryo development in the mutant was retarded relative to that in the wild type, and the former ultimately ceased at the globular stage (Table III; Fig. 8). This inh3 embryo showed a phenotype similar to that of the mutant, i.e. a defect in the control of cell division and the cell cycle, similar to that observed in rpn1a (Brukhin et al., 2005
Furthermore, recent characterizations of yeast Ypi1 led us to speculate that AtInh3 plays a role in the regulation of mitosis. Deletion of YPI1 is lethal in yeast, and conditional suppression of YPI1 results in the inhibition of cell growth of mid-mitosis (García-Gimeno et al., 2003
Plant Materials
Plants of Vicia faba ‘Ryosai Issun’ were cultured hydroponically in a greenhouse as described previously (Shimazaki et al., 1992
The yeast two-hybrid screen was performed using the MATCHMAKER Two-Hybrid System 2 (CLONTECH). The full-length VfPP1c-1 was fused in-frame to the GAL4 DNA-binding domain in the bait plasmid pAS2-1 (CLONTECH). A cDNA library was constructed from Vicia guard cell protoplasts into the phagemid vector pAD-GAL4-2.1 (Stratagene) as described previously (Emi et al., 2005
Full-length amino acid sequences were used for phylogenetic analyses. Sequence alignment was performed using the ClustalW program with default parameters (gap opening penalty, 10.00; gap extension penalty, 0.20; delay divergent cutoff, 30%; Supplemental Fig. S1). Phylogenetic analysis was done using MEGA software, version 4, with the neighbor-joining method (Tamura et al., 2007
3' RACE was done using a GeneRacer kit (Invitrogen) with two primers (5'-TCTCGTGGAAAGATGGCACTGTGGA-3' and 5'-TGAAGCTGGCCCGAGCAGTTAGGT-3'). Full-length VfINH3 was obtained by inverse PCR with circular cDNA prepared from the first-strand cDNA using T4 polynucleotide kinase and T4 RNA ligase (TaKaRa). The first PCR was carried out with the primers 5'-GCAAGAATCACGATGAAGCTG-3' and 5'-CTGCATGAACTCATTGTCCAC-3', and the nested PCR was performed with the primers 5'-CCGAGCAGTTAGGTTCTGATTG-3' and 5'-AGTGCCATCTTTCCACGAGAC-3'. The resulting PCR products were subcloned into a pCR4-TOPO vector (Invitrogen). Sequences were determined from both strands of the cDNA (ABI PRISM 3100; Applied Biosystems).
The bait and prey constructs were prepared using pAS2-1 and pACT2 (CLONTECH) and transformed into the yeast strain Y190. The β-galactosidase liquid culture assay was carried out using o-nitrophenyl β-D-galactopyranoside (ONPG) as a substrate. Briefly, overnight cultures grown at 30°C in synthetic complete media lacking Trp and Leu were centrifuged at 12,000g for 10 s, and the resulting pellet was washed with Z buffer (pH 7.0) containing 60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, and 1 mM MgSO4. The samples were resuspended in Z buffer and incubated for 5 min at 28°C. Thirty-two microliters of chloroform and 56 µL of 0.1% (w/v) SDS were added to 800 µL of the samples and further incubated for 5 min at 28°C. The assays were started by adding ONPG 160 µL of 0.4% (w/v) ONPG, and the reaction was performed for 120 min at 28°C. The reactions were stopped by adding 400 µL of 1 M Na2CO3. The samples were centrifuged at 10,000g for 10 min, and the OD420 of the supernatant was measured.
Site-directed mutagenesis was performed using a QuikChange site-directed mutagenesis kit (Stratagene). The primer sets used were 5'-AGAAGAAGAAGGTCTCGGCGAAAGATGGCACTGTGG-3' and 5'-CCACAGTGCCATCTTTCGCCGAGACCTTCTTCTTCT-3' for VfINH3-W59A, and 5'-GGAAGAAGAAGAAAGTTTCAGCGAAAGATGGGACTGTAGACA-3' and 5'-TGTCTACAGTCCCATCTTTCGCTGAAACTTTCTTCTTCTTCC-3' for AtINH3-W43A.
Full-length VfINH3 and AtINH3 were fused to a polyhistidine tag and thrombin cleavage sequence and subcloned into pFLAG-MAC expression vector (Sigma-Aldrich). E. coli strain BL21 was used as the host for protein expression. Cell growth and induction conditions were as described previously (Zhang et al., 1998
Full-length VfPP1c-1 and TOPP4 were subcloned into pGEX-2T (GE Healthcare), and the recombinant proteins were prepared as described previously (Takemiya et al., 2006
Pull-down assays were carried out using recombinant PP1c and Inh3. One microgram of GST fusion proteins (GST:VfPP1c-1 and GST:TOPP4) was allowed to bind to glutathione Sepharose 4B (GE Healthcare) for 1 h at 4°C with gentle shaking. The beads were incubated with E. coli lysate (20 µg protein) containing FLAG:VfInh3, FLAG:VfInh3-W59A, FLAG:AtInh3, or FLAG:AtInh3-W43A for 1 h at 4°C. The proteins on beads were solubilized and subjected to SDS-PAGE on 12.5% acrylamide gel. The proteins were immunodetected by anti-GST polyclonal (GE Healthcare) or anti-FLAG (Sigma-Aldrich) monoclonal antibodies.
The AtINH3 promoter region (2,151 bp) including the start codon was amplified by PCR from Arabidopsis genomic DNA using the primer pair of 5'-CCCAAGCTTAAGAACCAGAAAAATTAAACATTTACC-3' and 5'-CGGGATCCCATAGCTGAAGATTAGCTTTCAAAATCTG-3'. The fragment was subcloned into the HindIII and BamHI sites of pCAMBIA1300 binary vector (CAMBIA) with the nopaline synthase terminator. Full-length AtINH3 or AtINH3-W43A cDNA was also amplified using the primers 5'-CCCAAGCTTATGAGCACAGCAACAAGG-3' and 5'-CCCAAGCTTGGATCCTTAGTCAACGGCTTTAGAATC-3' and cloned into a pCRII vector (Invitrogen) containing the 3xFLAG sequence. The resulting 3xFLAG:AtINH3 fragment was introduced into the BamHI site of the pCAMBIA1300-AtINH3pro vector. The construct was transformed into Arabidopsis Col-0 plants using an Agrobacterium tumefaciens-mediated method.
Total proteins of 3-week-old Arabidopsis Col-0 and transgenic plants were extracted by homogenizing the seedlings (0.4 g) in extraction buffer containing 50 mM MOPS-KOH (pH 7.5), 2.5 mM EDTA, 100 mM NaCl, 2 mM phenylmethane sulfonyl fluoride, and 20 µM leupeptin. Triton X-100 was added to the extracts (1 mg protein) at 1% (w/v) and incubated at 4°C for 1 h. After centrifugation at 10,000g for 10 min, the supernatants were mixed with anti-FLAG M2 Agarose Affinity Gel (Sigma-Aldrich) and incubated at 4°C for 2 h. After washing four times, the proteins were eluted with 3xFLAG peptide (Sigma-Aldrich), solubilized, and subjected to SDS-PAGE on a 12.5% acrylamide gel. Proteins immunoprecipitated by anti-FLAG antibodies were visualized using the antibodies against PP1c, which were raised against the full-length TOPP4 prepared in E. coli. The 14-3-3 protein was immunodetected in the same supernatants as described previously (Kinoshita and Shimazaki, 1999
Full-length AtINH3 and AtINH3-W43A were subcloned into the CaMV35S
Total RNA was extracted from guard cell protoplasts, mesophyll cell protoplasts, leaves, stems, roots, green siliques, and flowers of V. faba and Arabidopsis using ISOGEN (Nippon Gene). The first-strand cDNA was synthesized from 1 µg of total RNA using a SuperScript III first-strand synthesis system (Invitrogen). The primer sets used for PCR were 5'-GATCCTCTTCATAAGAAATCGTGT-3' and 5'-AAACCGTGTTTTATTCCGGAATGT-3' for VfINH3; 5'-GTAGGTGATGAAGCCCAATC-3' and 5'-GGAATCCAACACAATACCAG-3' for V. faba ACTIN (VfACT); 5'-AGTTTCTCGATTCAACAAGCGTC-3' and 5'-TTAGTCAACGGCTTTAGAATCATTAG-3' for AtINH3; 5'-ACTTTACGCCAGTGGTCGTACAAC-3' and 5'-AAGGACTTCTGGGCACCTGAATCT-3' for ACT8; and 5'-CTCAAGAGGTTCTCAGCAGTA-3' and 5'-TCACCTTCTTCATCCGCAGTT-3' for β-TUBULIN (TUB), respectively. The PCR reaction mixture was denatured at 95°C for 2 min followed by 27 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min for VfACT, ACT8, and TUB or 30 cycles of the same protocol for VfINH3 and AtINH3.
The AtINH3 promoter region (2,148 bp) including the start codon was amplified by PCR from Arabidopsis genomic DNA using the primers 5'-CCCAAGCTTAAGAACCAGAAAAATTAAACATTTACC-3' and 5'-CATGCCATGGCTGAAGATTAGCTTTCAAAATCTG-3'. The fragment was subcloned into the HindIII and NcoI sites of the pCAMBIA1303 vector (CAMBIA), leading to a fusion between the AtINH3 promoter and GUS. The construct was transformed into Arabidopsis Col-0 plants using A. tumefaciens-mediated methods, and the resulting transformants were selected on agar plates with hygromycin. The GUS assay was done for homozygous T3 plants. The plant materials were incubated with GUS staining solution containing 100 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.1% (w/v) Triton X-100, and 0.5 mg mL–1 X-Gluc (5-bromo-4-chloro-3-indolyl-β-glucuronide) for 6 h at 37°C. Stained samples were fixed in the solution containing 85% (v/v) ethanol and 15% (v/v) acetic acid for 2 h at room temperature.
Wild-type and heterozygous mutant siliques were fixed in 90% (v/v) ethanol and 10% (v/v) acetic acid overnight, washed with a graded ethanol series (90%, 70%, 50%, and 30% ethanol) for 20 min each, and cleared in a derivative of Hoyer's solution (chloral hydrate/glycerol/water, 8 g:1 mL:2 mL) for 2 to 24 h at room temperature. Cleared seeds were observed using Digital Eclipse C1 (Nikon) equipped with differential interference contrast optics.
Leaves were homogenized in extraction buffer containing 0.1 M Tris, 2 M NaCl, 25 mM EDTA, and 2% (w/v) cetyltrimethylammonium bromide and incubated at 60°C for 30 min. After centrifugation at 10,000g for 5 min, the supernatant was mixed with chloroform and centrifuged again. The DNA in the aqueous phase was precipitated with 2-propanol, washed with 70% (v/v) ethanol, and used for PCR. Genotype analyses of inh3-1 and inh3-2 were conducted by PCR using the gene-specific primers S1 (5'-AGTTTCTCGATTCAACAAGCGTC-3') and AS1 (5'-TTAGTCAACGGCTTTAGAATCATTAG-3') and the T-DNA border primers LB1 (5'-TGGTTCACGTAGTGGGCCATCG-3') and LB2 (5'-GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC-3'). For complementation lines, a pair of gene-specific primers, S2 (5'-GTTTCTCGATTCAACAAGCGTCCC-3') and AS2 (5'-GAAGGAGTTAACATTGCAATGAATCTGC-3'), were used for PCR, and the resulting products were digested with BamHI.
For complementation of inh3-1, a 3,463-bp fragment of AtINH3 was amplified from Arabidopsis genomic DNA. First, a 2,151-bp fragment corresponding to the promoter region of AtINH3 was amplified using the primers 5'-CCCAAGCTTAAGAACCAGAAAAATTAAACATTTACC-3' and 5'-CGGGATCCCATAGCTGAAGATTAGCTTTCAAAATCTG-3', digested with HindIII and BamHI, and subcloned into pCAMBIA1300 binary vector (CAMBIA). The remaining 1,315-bp fragment including the coding sequence and the 3'-untranslated region of AtINH3 was amplified using the primers 5'-CGGGATCCATGAGCACAGCAACAAGGCCTTC-3' and 5'-CGGGATCCAGAGAGACCCATGATTATCCAGCC-3', digested with BamHI, and introduced into the pCAMBIA1300 with a 2,151-bp partial fragment. The resulting construct was used to transform heterozygous inh3-1 plants, and the transformants were selected on the agar plates supplemented with hygromycin.
For silencing of AtINH3 expression, full-length AtINH3 cDNA (321 bp) was amplified by PCR using the primer pairs (5'-GGGGTACCATGAGCACAGCAACAAGG-3' and 5'-CCGCTCGAGGATCCGTCAACGGCTTTAGAATCATTAG-3') and (5'-CCCAAGCTTATGAGCACAGCAACAAGG-3' and 5'-GCTCTAGAGGATCCGTCAACGGCTTTAGAATCATTAG-3') and subcloned in both the sense and antisense directions into a pKANNIBAL vector (Wesley et al., 2001 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers At2g31305 (AtINH3), AB372570 (VfINH3), NP_001052397 (OsINH3a), NP_001055058 (OsINH3b), and ABN09808 (MtINH3).
The following materials are available in the online version of this article.
We thank M. Tasaka and M. Furutani (Nara Institute of Science and Technology) for their valuable suggestions and N. Nishihara and M. Inoue in our laboratory for their helpful technical assistance. We are also grateful to the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants and the ABRC at Ohio State University for providing the mutant seeds. Received January 6, 2009; accepted March 24, 2009; published March 27, 2009.
1 This work was supported by the Japanese Ministry of Education, Science, Sports and Culture, a Grant-in-Aid for Scientific Research on Priority Areas (grant no. 17084005), and a Grant-in-Aid for Scientific Research (B) (grant no. 19370020) to K.S.  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: Ken-ichiro Shimazaki (kenrcb{at}mbox.nc.kyushu-u.ac.jp).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.135335 * Corresponding author; e-mail kenrcb{at}mbox.nc.kyushu-u.ac.jp.
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N:DsRed; Pollmann et al., 2006
-sGFP(S65T)-nos3' (Niwa et al., 1999
1 and PP1
, respectively. Arch Biochem Biophys 443: 33–44