Plant and Cell Physiology Advance Access originally published online on January 28, 2009
Plant and Cell Physiology 2009 50(3):429-438; doi:10.1093/pcp/pcp012
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Rapid Paper |
The 14-3-3 Protein GF14c Acts as a Negative Regulator of Flowering in Rice by Interacting with the Florigen Hd3a
Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0101 Japan
*Corresponding author: E-mail, simamoto{at}bs.naist.jp; Fax, +81-743-72-5502.
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Abstract |
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Hd3a and FT proteins have recently been proposed to act as florigens in rice and Arabidopsis, respectively; however, the molecular mechanisms of their function remain to be determined. In this study, we identified GF14c (a 14-3-3 protein) as an Hd3a-interacting protein in a yeast two-hybrid screen. In vitro and in vivo experiments, using a combination of pull-down assays and bimolecular fluorescence complementation, confirmed the interaction between Hd3a and GF14c. Functional analysis using either GF14c overexpression or knockout transgenic rice plants indicated that this interaction plays a role in the regulation of flowering. GF14c-overexpressing plants exhibited a delay in flowering and the knockout mutants displayed early flowering relative to the wild-type plants under short-day conditions. These results suggest that GF14c acts as a negative regulator of flowering by interacting with Hd3a. Since the 14-3-3 protein has been shown to interact with FT protein in tomato and Arabidopsis, our results in rice provide important findings about FT signaling in plants.
Keywords: Flowering - GF14c - Hd3a - Protein interaction - Rice.
Abbreviations: 3-AT, 3-aminotriazole; BiFC, bimolecular fluorescence complementation; bZIP, basic/leucine zipper; CaMV, cauliflower mosaic virus; DTT, dithiothreitol; GFP, green fluorescent protein; GST, glutathione S-transferase; GUS, β-glucuronidase; LD, long day; PEBP, phosphatidyl-ethanolamine-binding protein; QTL, quantitative trait locus; RKIP, Raf kinase inhibitor protein; RNAi, RNA interference; RT–PCR, reverse transcription–PCR; SAM, shoot apical meristem; SD, short day; TBS, Tris-buffered saline; ZT, zeitgeber time.
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsSupplementary dataFundingAcknowledgmentsReferences |
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Flowering is critical for growth and reproduction in plants, and is controlled by both environmental and endogenous conditions. One of the most important factors that controls flowering is the plant's response to photoperiod (Imaizumi and Kay 2006
). In addition to the photoperiodic pathway, the regulation of flowering time involves complex signaling pathways, such as the vernalization, autonomous flowering and gibberellins pathways. Arabidopsis thaliana and Oryza sativa are used as models to study the regulation of flowering time in long-day (LD) plants and short-day (SD) plants, respectively. Three genes that constitute the major genetic pathway in the photoperiodic regulation of flowering in rice have been isolated: OsGI (O. sativa GIGANTEA), an ortholog of Arabidopsis GI; Hd1 (Heading date 1), an ortholog of Arabidopsis CO (CONSTANS); and Hd3a (Heading date 3a), an ortholog of Arabidopsis FT (FLOWERING LOCUS T). The major difference between rice, an SD plant, and Arabidopsis, an LD plant, is the regulation of Hd3a/FT by Hd1/CO. Under LD conditions, this regulation is positive in Arabidopsis but negative in rice (Hayama et al. 2003, Izawa 2007, Tsuji et al. 2008).
Hd3a was first identified as a quantitative trait locus (QTL) that promotes flowering of rice under SD conditions (Yamamoto et al. 1998, Monna et al. 2002). Overexpression of Hd3a protein with a constitutive promoter (Kojima et al. 2002) or vascular-specific promoters (Tamaki et al. 2007) results in an early-flowering phenotype, and suppression of Hd3a with RNA interference (RNAi) delays flowering (Komiya et al. 2008). Hd3a is a member of a large gene family consisting of at least 13 genes in the rice genome (Chardon and Damerval 2005), and at least two paralogs, RFT1 (Rice FLOWERING LOCUS T1) and FTL (FT-Like), are reported to have a flowering promotion function (Izawa et al. 2002, Komiya et al. 2008).
Hd3a expression is regulated by Hd1 and Ehd1 (Early heading date1) that encodes a B-type response regulator and is a unique flowering time gene in rice (Doi et al. 2004). Ehd1 promotes floral transition preferentially under SD conditions, even in the absence of functional alleles of Hd1. Expression analysis revealed that Ehd1 functions upstream of Hd3a, RFT1 and some MADS-box genes (Doi et al. 2004). More recently, Ghd7, which encodes a CCT- (CO, CO-LIKE and TIMING OF CAB1) domain protein, was isolated based on natural variation in rice (Xue et al. 2008). Ghd7 affects levels of Ehd1 and Hd3a transcripts, but does not affect Hd1 mRNA levels. Ghd7 represses Ehd1 and Hd3a expression under LD conditions, leading to delayed flowering. Therefore, two independent floral pathways are present in rice: the conserved Hd1 pathway and a unique Ehd1 pathway that may integrate environmental photoperiod signals into the expression of FT-like genes (Izawa 2007, Tsuji et al. 2008). Recently, another flowering gene, RID1/OsId1/Ehd2, was identified in rice. This gene encodes a putative transcription factor with a zinc finger motif orthologous to ID1 (INDETERMINATE1) that promotes flowering in maize (Zea mays) (Matsubara et al. 2008, Park et al. 2008, Wu et al. 2008). RID1/OsId1/Ehd2 promotes the floral transition mainly by up-regulating Ehd1 and genes downstream of Ehd1, such as Hd3a and RFT1.
FT encodes an approximately 23 kDa protein whose sequence is similar to that of Raf kinase inhibitor protein (RKIP) and phosphatidylethanolamine-binding protein (PEBP) (Ahn et al. 2006). The PEBP family regulates signaling pathways to control growth and differentiation. PEBPs seem to act biochemically as inhibitors, binding signaling components to reduce the flux through their pathways (Hanzawa et al. 2005). The crystal structures of PEBP from human and bovine sources (Banfield et al. 1998) and CENTRORADIALIS from Antirrhinum (Banfield and Brady 2000), as well as those of Arabidopsis [TERMINAL FLOWER1 (TFL1) and FT] (Ahn et al. 2006), indicate that the ligand-binding pocket is accessible for interacting with protein partners. PEBPs may also act as either scaffolds for or regulators of signaling complexes, as suggested by the finding that SP (SELF PRUNING) and SFT (SINGLE-FLOWER TRUSS), the tomato orthologs of TFL1 and FT, respectively, can interact with diverse proteins (Pnueli et al. 2001, Lifschitz et al. 2006). In Arabidopsis, FT interacts with the basic/leucine zipper (bZIP) transcription factor FD (Abe et al. 2005, Wigge et al. 2005). The bZIP transcription factor SPGB in tomato, a homolog of FD, also interacts with SP and SFT. Structural analysis of CEN indicates that its ligand-binding pocket is incapable of accommodating phosphoryl groups; however, PEBPs may mediate signaling via their association with phosphorylated proteins. Indeed, a mouse PEBP, RKIP, has been shown to interact with and inhibit the activity of the kinase Raf1 (Yeung et al. 1999).
Most recently, Hd3a and its ortholog have been proposed as florigens, or mobile flowering signals (Corbesier et al. 2007, Jaeger and Wigge et al. 2007, Lin et al. 2007, Mathieu et al. 2007, Tamaki et al. 2007, Notaguchi et al. 2008, Tsuji et al. 2008). The next question that needs to be addressed is the mechanism of Hd3a function. One important step in the characterization of Hd3a function is to identify other proteins with which it interacts.
A study in tomato revealed several SP-interacting proteins including a 14-3-3 family member, a protein kinase and a bZIP transcription factor (Pnueli et al. 2001). SFT also interacts with 14-3-3, as well as with bZIP (Pnueli et al. 2001). In Arabidopsis, FT interacts with FD and 14-3-3 proteins (Abe et al. 2005, Pnueli et al. 2001, Wigge et al. 2005); however, no Hd3a-interacting proteins have yet been identified in rice.
In this study, we identified GF14c (a G-box factor 14-3-3c protein) as an Hd3a-interacting protein both in vitro and in vivo. Since 14-3-3 family members interact with diverse proteins, it is of interest to understand their role in Hd3a signaling. Functional analyses using both knockout and overexpressing plants indicate that GF14c acts as a negative regulator of flowering in rice.
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Results |
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Hd3a interacts with GF14c in a yeast two-hybrid system
To understand the molecular function of Hd3a, we perfor-med a yeast two-hybrid screen to search for Hd3a-interacting proteins, using full-length Hd3a as bait for a cDNA library constructed from leaf blades harvested when Hd3a expression was induced. From 1.6x106 yeast transformants, 96 clones grew on selective medium lacking histidine. Three independent clones were identified as GF14c, an isoform of the rice 14-3-3 gene family (Table 1). Only a partial fragment in the C-terminal region (115–256) was identified originally from the yeast-two hybrid screen. An experiment using full-length Hd3a and full-length GF14c, either as bait or as prey, further confirmed that Hd3a and GF14c interact in the yeast system (Fig. 1). In plants, 14-3-3 proteins have been known to interact with Hd3a orthologs: FT in Arabidopsis and SP and SFT in tomato (Pnueli et al. 2001
). GF14 family members have roles in both disease resistance and development in rice (Cooper et al. 2003). We are interested in further characterizing the molecular function of GF14c in association with Hd3a throughout rice development, particularly during the floral transition.
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Tissue-specific expression of Hd3a and GF14s in rice
From the International Rice Annotation Project Database (Rice Annotation Project, 2008
), we identified GF14 family members. GF14s belong to the 14-3-3 protein group and have eight isoforms, designated as GF14a–h in rice (Chen et al. 2006a, Yao, et al. 2007), that share 85–95% amino acid identity (Fig. 2A). To determine whether the GF14 genes are expressed in rice organs, we performed semi-quantitative reverse transcription–PCR (RT–PCR) using specific primers (Fig. 2B). The GF14 genes were expressed in all tested organs, suggesting that they have broad functions in rice growth and development. The expression levels of GF14 a–f seem to be higher than those of GF14g and GF14h. No diurnal changes or developmental patterns of GF14c expression were observed (Supplementary Fig. S1), indicating that GF14c is expressed independently of the photoperiod and abundantly during plant development. We also examined the distribution of Hd3a and 14-3-3 proteins in rice plants. Total proteins from Hd3a-overexpressing plants under its native promoter (Hd3a::Hd3a:GFP) (Tamaki et al. 2007) and non-transgenic control plants co-expressed Hd3a and 14-3-3 proteins in both leaf blades and stems (Fig. 2C).
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Subcellular localization of Hd3a and GF14c
To gain insight into the molecular function of Hd3a and GF14c, we made a fusion construct to express mCherry fluorescent protein-linked Hd3a under the ubiquitin promoter and green fluorescent protein (GFP)-linked GF14c driven by the caulifower mosaic virus (CaMV) 35S promoter, and analyzed the intracellular localization of Hd3a and GF14c. These constructs were introduced into rice protoplasts isolated from rice suspension culture cells. All rice protoplasts observed in this experiment showed that Hd3a–mCherry was associated with both the cytoplasm and nucleus; however GFP–GF14c predominantly localized in the cytoplasm (75% of rice protoplasts observed) (Fig. 2D). The predominant cytoplasmic localization of 14-3-3 proteins may suggest that they function as cytoplasmic anchors that regulate the import of proteins into the nucleus or other organelles or promote export from the nucleus to the cytoplasm (Muslin and Xing 2000
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In vitro interaction of Hd3a and GF14c
To study the interaction between Hd3a and GF14c, we performed a glutathione S-transferase (GST) pull-down assay. A GST–Hd3a fusion protein was pulled down with His-tagged GF14c, as shown in Fig. 3A, indicating that Hd3a interacted with GF14c in vitro. Results of this experiment were thus consistent with the results of the yeast two-hybrid experiment.
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-His antibody. (B) BiFC analysis of the interaction between Hd3a and GF14c in rice protoplasts. Bars = 10 µm.The Hd3a and GF14c complex is localized in the cytoplasm by BiFC
We used bimolecular fluorescence complementation (BiFC) to determine the distribution of Hd3a and GF14c in vivo. The expression vectors for Hd3a protein fused to the N-terminal half of mVenus (Vn) and GF14c protein fused to the C-terminal half of mVenus (Vc) were transiently introduced into the rice protoplasts, with the mCherry expression plasmid as a marker for transformed cells. As shown in Fig. 3B, 95% of transformed rice protoplasts observed in this experiment showed Hd3a and GF14c interaction in both protein fusion combinations as documented by a strong green fluorescence concentrated in the cytoplasm. Hd3a-Vn/GUS-Vc or GUS-Vn/GF14c-Vc combination showed remarkably few numbers of cells with Venus fluorescence (0–10% of the transformed cells). These results clearly demonstrate that Hd3a interacts with GF14c mainly in the cytoplasm.
GF14c-overexpressing and gf14c mutant plants
To characterize the function of GF14c, a GF14c overexpression (ox) construct driven by the CaMV 35S promoter was generated and transformed into rice plants (Fig. 4A). T1 generation transgenic plants (n = 15) were used for the phenotypic analysis of GF14c. GF14c-ox plants were delayed by 5–20 d in flowering relative to wild-type plants (Fig. 4B, C). A t-test confirmed that the flowering time was significantly different between GF14c-ox and wild-type plants (P = 0.0046). Other phenotypes observed in this mutant were erect leaves and longer culms compared with the wild-type plants (data not shown). In GF14c-ox plants, overexpression of GF14c at both the mRNA and the protein level was confirmed (Fig. 4D, E).
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To study further the function of GF14c in flowering, we also used the gf14c/GF14c heterozygous T-DNA insertion mutants in japonica rice cv ‘Dongjin’ generated by Postech, Korea. One heterozygous line (Fig. 5B) possessed a T-DNA insertion in the second exon of GF14c (Fig. 5A, D). We germinated 50 seeds derived from the gf14c T-DNA mutants and found that 24 were heterozygous, seven were wild type and 19 did not germinate, which were likely to be homozygous, suggesting that the mutation may be lethal. The gf14c/GF14c plants flowered earlier than those of the wild type by about 14 d (12–17-d depending on the plants) (Fig. 5C). A t-test confirmed that the flowering time was significantly different between the wild type and gf14c (P = 0.0387, n = 5). GF14c expression at both the mRNA and protein levels in these mutants plants was lower than in wild-type plants (Fig. 5E, F). These results clearly indicate that GF14c is involved in regulation of flowering time in rice.
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Discussion |
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Hd3a is thought to move along leaf cells and the vascular system of leaves and stems, and exerts its action in the shoot apical meristem (SAM). Since the tissue localization of Hd3a protein was determined using transgenic rice plants expressing Hd3a–GFP fusion protein driven by the Hd3a promoter, and Hd3a protein was present in vascular tissue from the leaf blades and transported through the phloem (Tamaki et al. 2007
), we prepared a cDNA library from leaf blades to search for Hd3a-interacting proteins in a yeast two-hybrid screen. We identified GF14c, a member of the 14-3-3 protein family whose members are highly conserved in all eukaryotes (Ferl 1996). The 14-3-3 proteins are well known as proteins that are involved in signaling pathways in eukaryotes. In plants, 14-3-3 proteins have been shown to interact with FT and TFL1 in Arabidopsis, and with SP and SFT in tomato (Pnueli et al. 2001, Lifschitz et al. 2006). More recently, a comprehensive study using yeast two-hybrid and affinity chromatography with mass spectrometry provided a large number of novel putative 14-3-3-interacting partners in barley (Schoonheim et al. 2007). Interestingly, a wide range of 14-3-3 targets have functions in various signal transduction pathways and might form a platform for cross-talk in the signal transduction pathway. Binding of 14-3-3 proteins regulates their partner proteins through a variety of mechanisms, such as altering their catalytic activity, subcellular localization, stability or their interaction with other proteins (Roberts, 2003). Identification of GF14c as an Hd3a partner has been confirmed by several methods. The results from yeast two-hybrid assays using a full-length construct, in vitro pull-down and BiFC assays demonstrated the interaction between Hd3a and GF14c in rice. The subcellular distribution of GF14c demonstrated their localization in both the cytoplasm and nucleus. The 14-3-3 proteins are highly conserved throughout the animal and plant kingdom. In Arabidopsis, there are at least 12 isoforms, whereas eight isoforms exist in rice. These proteins are known as a component associated with a number of different proteins in signal transduction pathways, including plant hormone signaling pathways.
Overexpression of GF14c caused late flowering, suggesting that 14-3-3 functions as a negative regulator of flowering. Hd3a protein is localized in both the cytoplasm and nucleus; however, GF14c is mainly localized in the cytoplasm and only a very weak fluorescent signal was observed in the nucleus (Fig. 2D). To address the question of whether the interaction between Hd3a and GF14c would inhibit shuttling of Hd3a from the cytoplasm into the nucleus, we performed a BiFC experiment. The results demonstrate that the Hd3a–GF14c protein complex is mainly localized in the cytoplasm (Fig. 3B). This result strongly suggests that Hd3a and GF14c interaction increases Hd3a cytoplasmic retention. Therefore, the delayed flowering phenotype in GF14c-ox plants can be explained by the increased cytoplasmic retention of Hd3a by GF14c (Fig. 6). In Arabidopsis, to initiate floral transition in the shoot apex, FT interacts with FD, a bZIP transcription factor that localizes in the nucleus to induce target meristem identity genes such as APETALA1 (AP1) (Abe et al. 2005). This process could be attenuated by the cytoplasmic retention of Hd3a by GF14c. Several lines of evidence indicate the function of 14-3-3 protein in nuclear–cytoplasmic shuttling in the signal transduction pathway (Igarashi et al. 2001, Ishida et al. 2004).
Another mechanism which could possibly explain the phenotypes of GF14c-ox plants and the gf14c knockout mutants is the interaction of Hd3a with GF14c, which inhibits movement of Hd3a in the long-distance trafficking through the phloem (Fig. 6). Interestingly, the gf14c knockout mutants exhibited an early flowering phenotype. These mutants also showed a dwarf phenotype and increased tiller numbers (Supplementary Fig. S2), suggesting that GF14c could also function independently of flower induction at the SAM. When expression levels of GF14c are low in the leaf, Hd3a protein may be capable of moving long distances freely from companion cells to the phloem sieve elements since Hd3a is a small protein that is under the size exclusion limit of plasmodesmata (Lough and Lucas 2006, Giakountis and Coupland 2008). In contrast to our results in rice, in Arabidopsis, loss of 14-3-3 µ and 14-3-3 
functions produced a slight delay in flowering (3–5 d) in comparison with the wild type (Mayfield et al. 2007). These authorts also tested the interaction with components of the photoperiodic pathway, such as CO, GI, ZTL, PIF3 and TOC1; however, the interaction with FT was not tested. This previous study also showed that 14-3-3 µ and 14-3-3 interacted with CO, a central regulator of the photoperiod pathway (Mayfield et al. 2007). Therefore, although our study demonstrates an interaction between Hd3a and GF14c, and indicates that GF14c acts as a negative regulator of flowering in rice, molecular mechanisms of GF14c function in floral transition and development remain to be studied.
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Materials and Methods |
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Plant materials and growth conditions
Japonica rice cultivars ‘Norin 8’ (N8) and ‘Dongjin’ were used as wild-type plants. GF14c-overexpressing plants (35S::GF14c) were generated by Agrobacterium-mediated transformation in an N8 background. T-DNA insertion alleles of GF14c in a ‘Dongjin’ background (IB-10131) were obtained from Dr. Gynheung An, POSTECH, Korea. To confirm insertion sites, the sequences flanking the right border of T-DNA were determined using primers designed from sequence information provided in the POSTECH database. To determine flowering time phenotypes, all plants were grown in climate chambers under SD conditions with a 24 h temperature cycle (10 h, 30°C during subjective day; 14 h, 25°C during subjective night). The humidity was 70%. The fluence of light was
300 µmol–2 s–1 (400–750 nm) under SD conditions.
cDNA library and yeast two-hybrid screening
Poly(A)+ RNA was isolated from wild- type N8 rice leaf blades, harvested at 35–40 d after sowing at zeitberger time (ZT) 0, 2 and 4, with an RNA Easy Prep kit (TAKARA SHUZO CO. LTD., Kyoto, Japan) and used to construct a cDNA library with a cDNA synthesis kit (Stratagene, La Jolla, CA, USA). The resultant cDNAs were inserted into the pVP16 vector and introduced into Saccharomyces cerevisiae L40 cells. A full-length Hd3a bait was cloned into the pBTM116 vector. Independent clones (1.6x106) were screened for interaction with Hd3a. A swapping experiment, using full-length constructs of GF14c and Hd3a, either as bait or as prey, was performed. Interactions were tested on SC medium lacking histidine (–H) or lacking histidine and containing 2.5 mM 3-aminotriazole (3-AT).
Full-length cDNA construction
Since only a partial cDNA was obtained from yeast two-hybrid screening, we constructed a full-length GF14c cDNA based on sequence information in the rice DNA database. We designed forward and reverse primers (see Supplementary Table S1) to produce a full-length PCR fragment using cDNA as a template. The PCR fragment was then subcloned into the pENTR/D-TOPO cloning vector (Invitrogen, Carlsbad, CA, USA) to obtain an entry clone.
Database analysis of GF14c
Rice GF14 family sequences were obtained from the GenBank database and the International Rice Annotation Project Database (Rice Annotation Project, 2008). For data mining of GF14c, the deduced amino acid sequences were aligned using ClustalW with default parameters, and a phylogenetic tree was generated using the neighbor-joining method.
In vitro pull-down assay
Hd3a and GF14c were cloned into pDEST15 and pDEST17 vectors (Invitrogen), respectively. GST, GST–Hd3a and His–GF14c were expressed in Escherichia coli BL21 (DE3) (Invitrogen) and purified with glutathione–agarose (Sigma, Tokyo, Japan) and HisTrap (Amersham) columns according to the manufacturers’ instructions. In vitro binding assays were performed as follows. The concentration of each fusion protein was determined by Coomassie staining. Equal amounts of GST–Hd3a protein coupled to glutathione–Sepharose 4B beads and His–GF14c were incubated in TEDM buffer [10 mM Tris–HCl, pH 7.5/0.5 mM EDTA, pH 7.5/1 mM dithiothreitol (DTT) and 1 x Complete Proteinase Inhibitor Cocktail (Roche, Mannheim, Germany)]. The beads were then washed four times with binding buffer. Bound proteins were eluted in 1 x SDS sample buffer by boiling for 5 min, separated by 10% SDS–PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Tokyo, Japan) and subjected to immunoblotting with anti-His antibody (BD Biosciences Pharmingen). After washing with Tris-buffered saline (TBS) containing 0.1% Tween (TBST), the membranes were incubated for 1 h with anti-mouse IgG conjugated to horseradish peroxidase (GE Healthcare). Detection was performed using enhanced chemiluminescence (ECL) protein gel blot detection reagents (GE Healthcare) and visualized using an LAS-1000 Imager (Fujifilm, Tokyo, Japan).
Tissue-specific expression
Total RNA from seeds, seedlings, leaf blades, leaf sheaths, stems, shoot apices, roots and rice suspension culture cells were extracted using TRIZOL reagent (Invitrogen) and treated with DNase I (Invitrogen). The cDNA was synthesized from 1 µg of total RNA using SuperScript II reverse transcriptase (Invitrogen). The cDNA was subjected to semi-quantitative analysis of gene expression using PCR with primers specific for GF14 family members, Hd3a and ubiquitin (see Supplementary Table S1).
GF14 protein expression in planta
Leaves and stems from wild-type, Hd3a::Hd3a:GFP-overexpressing, GF14c-overexpressing and gf14c knockout plants were harvested and ground to a fine powder in liquid nitrogen. Proteins were extracted as described previously (Mathieu et al. 2007), using an extraction buffer containing 150 mM NaCl, 50 mM Tris pH 7.5, 0.1% Tween 20, 10% glycerol, 1 mM DTT, 1 mM Pefabloc (Roche), 1x Halt Phosphatase Inhibitor Cocktail (Pierce) and 1x Complete Proteinase Inhibitor Cocktail (Roche). The extracts were centrifuged at 15,000 r.p.m. for 40 min at 4°C. The supernatant was separated by 12.5% SDS–PAGE and subjected to immunoblotting using the primary antibody (polyclonal rabbit anti-GFP antibody, Abcam) as described previously (Mathieu et al. 2007) and anti-14-3-3 antibody as described above. After washing with TBST the membranes were incubated for 1 h with anti-rabbit and anti-rat IgG conjugated to horseradish peroxidase, respectively (GE Healthcare). Detection was performed as above.
Subcellular localization
The GFP sequence was fused to either the C- or the N-terminus of GF14c using the Gateway system (Invitrogen). Expression of GF14c-tagged GFP constructs and Hd3a-tagged mCherry was driven by the CaMV 35S promoter and the ubiquitin promoter, respectively. Protoplast isolation from rice Oc suspension culture (Kyozuka and Shimamoto 1991) and protoplast transfomation (Chen et al. 2006b) were performed as described previously. After a 24 h incubation at 30°C, the protoplasts were examined under a confocal microscope (LSM510, Zeiss).
Bimolecular fluorescence complementation
For constructs used in the BiFC experimet, vectors containing the N- and C-terminal halves of the mVenus were kindly provided by S. Takayama, NAIST. Hd3a, GF14c and β-glucuronidase (GUS)-coding regions were cloned into the BiFC vectors and purified by the Purelink Plasmid Midiprep Kit (Invitrogen). The purified BiFC plasmids were introduced into the rice protoplasts as described previously (Kyozuka and Shimamoto 1991, Chen et al. 2006b). mCherry expression plasmid was introduced at the same time as the marker for transformed cells. The number of cells with reproduced mVenus fluorescence in the mCherry-expressing cells was scored.
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Supplementary data |
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Supplementary data are available at PCP online.
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Funding |
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The Ministry of Education, Culture, Sports, Science and Technology, Japan Grants-in-Aid for Scientific Research on Priority Areas (grants 10182102 and 19090013 to K.S.); the Japan Educational Exchanges and Services Iida International Student Scholarship (to Y.A.P.).
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Acknowledgments |
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We thank Shuji Yokoi for help in the early part of this work, Yukiko Konomi for technical assistance with transgenic plants, Gynheung An for providing gf14c T-DNA insertion mutants, Seiji Takayama for providing the BiFC vectors, and all members of the Shimamoto lab for helpful discussions.
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References |
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(Received December 26, 2008; Accepted January 21, 2009)
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