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First published online February 2, 2007; 10.1104/pp.106.095356 Plant Physiology 143:1293-1305 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
A Novel Ankyrin-Repeat Membrane Protein, IGN1, Is Required for Persistence of Nitrogen-Fixing Symbiosis in Root Nodules of Lotus japonicus1,[OA]National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305–8602, Japan (H.K., T.H., Y.U., H.K.); and Kazusa DNA Research Institute, Kisarazu, Chiba 292–0812, Japan (S.S., T.K., S.T.)
Nitrogen-fixing symbiosis of legume plants with Rhizobium bacteria is established through complex interactions between two symbiotic partners. Similar to the mutual recognition and interactions at the initial stages of symbiosis, nitrogen fixation activity of rhizobia inside root nodules of the host legume is also controlled by specific interactions during later stages of nodule development. We isolated a novel Fix– mutant, ineffective greenish nodules 1 (ign1), of Lotus japonicus, which forms apparently normal nodules containing endosymbiotic bacteria, but does not develop nitrogen fixation activity. Map-based cloning of the mutated gene allowed us to identify the IGN1 gene, which encodes a novel ankyrin-repeat protein with transmembrane regions. IGN1 expression was detected in all organs of L. japonicus and not enhanced in the nodulation process. Immunoanalysis, together with expression analysis of a green fluorescent protein-IGN1 fusion construct, demonstrated localization of the IGN1 protein in the plasma membrane. The ign1 nodules showed extremely rapid premature senescence. Irregularly enlarged symbiosomes with multiple bacteroids were observed at early stages (8–9 d post inoculation) of nodule formation, followed by disruption of the symbiosomes and disintegration of nodule infected cell cytoplasm with aggregation of the bacteroids. Although the exact biochemical functions of the IGN1 gene are still to be elucidated, these results indicate that IGN1 is required for differentiation and/or persistence of bacteroids and symbiosomes, thus being essential for functional symbiosis.
Legume plants form nitrogen-fixing root nodules in symbiotic associations with soilborne bacteria generally referred to as Rhizobium. The nodulation process of the host legumes is triggered by specific lipochitin-oligosaccharide signal molecules, Nod factors, secreted by rhizobia. Nod factors elicit a series of host responses, such as root hair deformation, preinfection thread (cytoplasmic bridge) formation, and cortical cell division leading to formation of nodule primordia (Spaink and Lugtenberg, 1994  ; D'Haeze and Holsters, 2002). Subsequently, rhizobia invade the root cortical cells through infection threads and are released into the host cell cytoplasm. The released bacteria reside in the nodule cells as enclosed by peribacteroid membrane (PBM) derived from the host plasma membrane to form a symbiotic organelle symbiosome. Finally, the rhizobia differentiate into the symbiotic form, bacteroids, and induce nitrogen-fixing activity.
These successive developmental stages of symbiotic nodules are established through highly coordinated actions of the genes of both symbiotic partners. Recent comprehensive analyses using expressed sequence tags accumulated from some legume species revealed that the nodulation process involves global and drastic changes in gene expression in the host legumes (Colebatch et al., 2002
To identify the host plant genes essential for nodulation process, extensive and systematic efforts have been made during the past decade to isolate various kinds of symbiosis mutants from two model legume species, Lotus japonicus and Medicago truncatula (Schauser et al., 1998
Hist– mutants represent genetic loci that have an effect on the infection process accompanied by cooperative histogenesis of nodule structures. The mutants of this category are characterized by defects in infection thread formation and/or its growth, as well as incomplete nodule organogenesis with absence of bacterial infected cells. L. japonicus alb1 (Imaizumi-Anraku et al., 1997
In contrast, genetic loci in Fix– mutants are attributed to much later stages of symbiotic nodule development, which involves the differentiation of rhizobia to bacteroids followed by induction of nitrogenase, and organization of metabolic functions required for nitrogen fixation in the host nodule cells. Fix– mutants form morphologically normal nodules with infected cells containing endosymbiotic bacteria, but exhibit no or very low nitrogen-fixing activity (Imaizumi-Anraku et al., 1997 We describe here the identification of a novel Fix– mutant designated ineffective greenish nodules 1 (ign1) of the model legume L. japonicus and molecular cloning of the IGN1 gene for the mutated locus by means of a map-based cloning strategy. On the basis of the mutant phenotype and structure of the gene identified, we discuss possible functions of the IGN1 gene in the establishment of functional symbiosis.
The ign1 Mutant Forms Ineffective Nodules A Fix– mutant, ign1, was isolated by somatic mutation through intensive culture of hypocotyl-derived calli followed by regeneration of the plants (see "Materials and Methods"). Segregation analysis of the F2 progeny after the cross with the wild types Gifu and MG-20 indicated that the ign1 locus is a monogenic recessive (data not shown). The ign1 mutant displayed typical nitrogen deficiency symptoms, such as chlorotic leaves, pigmented stems, and stunted shoot growth, when grown in nitrogen-free medium following inoculation of M. loti (Fig. 1, A and D ). At 18 d after inoculation with M. loti, nodules formed on the ign1 mutant were slightly smaller than those of wild-type Gifu plants and were white to pale green in color, whereas wild-type nodules were red because of the presence of abundant leghemoglobin in the infection zone (Fig. 1, B and C). Nitrogenase activity of the ign1 nodules as measured by acetylene reduction activity was very low as compared with wild-type nodules (Fig. 1E). Immunoblot analysis for nitrogenase proteins from 14-d-old nodules indicated that the component I and II proteins are both present in the bacteroids in ign1 nodules at the level slightly lower than those in wild-type nodules (data not shown).
There was no significant difference either in the nodule number per plant (Fig. 2B ) or the time course of nodulation between the ign1 mutant and wild-type Gifu plants (data not shown). When the plants were grown with sufficient supply of exogenous nitrogen, the ign1 mutants retained normal growth that is comparable to wild-type plants (Fig. 2A), indicating that the stunted growth of the mutants under symbiotic conditions is simply due to a defect in the nitrogen-fixing ability of the nodules. In addition, nodulation response of the mutants to increasing concentrations of supplemental nitrate was the same as wild-type plants (Fig. 2B). This indicates that nodulation of the ign1 mutants is equally sensitive to exogenous nitrate as that of the wild-type plants. There was no apparent abnormality in the morphology of leaves, stems, and roots of the mutants when grown under nonsymbiotic conditions.
Infected Cells in the ign1 Nodules Collapse Prematurely
In the wild-type L. japonicus Gifu, nodules are first visible as small bumps around 4 d post inoculation (dpi) and nitrogen fixation is initiated at 10 to 12 d (Kouchi et al., 2004
To examine the mutant nodule phenotype in more detail, ultrathin sections of the nodules at 8 to 13 dpi were observed by transmission electron microscopy (Fig. 4 ). Distinctive features of the infected cells of the ign1 nodules were evident at the ultrastructural level even at such early developmental stages. In wild-type Gifu nodules, infected cells were filled with PBM-enclosed bacteroids (symbiosomes; Fig. 4, A and B). Most of the symbiosomes were spherical and the PBM was adjacent to bacteroids. Some symbiosomes appeared to fuse with each other to form larger symbiosomes containing more than two bacteroids. The most striking difference in infected cells of the ign1 nodules as compared with those of wild-type nodules appeared on the symbiosome structures. Symbiosomes in the infected cells of ign1 nodules were irregularly shaped, enlarged relative to the bacteroids inside, and as a consequence the symbiosome space between the PBM and bacteroids increased in size. The abnormal symbiosome structures were clearly observed in 8 dpi nodules (Fig. 4C) and became more severe along the nodule age (Fig. 4E). Occasionally these structures seemed to be similar to lytic vacuoles as described recently for the prematurely senesced nodules of the sst1 (sulfate transporter) mutant of L. japonicus (Krusell et al., 2005 ). Bacteroids in the ign1 nodules appeared to be smaller in size and less dense than those of the wild type, suggesting that the bacteroid differentiation is inhibited and/or at least delayed in the ign1 nodules. Premature senescence is more or less a common feature of ineffective nodules formed on Fix– mutants. Abnormal enlargement of symbiosomes has been described as an early symptom of the premature senescence in the nodules of some Fix– mutants (Suganuma et al., 2003; Krusell et al., 2005; Hossain et al., 2006). To examine the possibility if the ineffectiveness (lack of nitrogen fixation) causes such abnormal symbiosome structures, we observed ultrastructures of the infected cells of the ineffective nodules formed by a nifH mutant ( nifH) of M. loti that lacks completely nitrogenase expression (Fig. 4F). The infected cell structures of nifH nodules are almost comparable to those of wild-type nodules at least during early stages of nodule development.
Map-Based Cloning of the IGN1 Gene
To identify the gene affected in the ign1 mutant, we employed a map-based cloning strategy using L. japonicus accession MG-20 Miyakojima as a crossing partner (Kawaguchi et al., 2001
Since we isolated only a single allele of the ign1 locus, it was imperative to test the complementation of the mutant phenotype with the candidate gene. A DNA fragment of 8.6-kb length was excised from a genomic library of L. japonicus Gifu plants, which contains the entire ORF of the candidate gene together with the 1.9-kb upstream and 3.5-kb downstream sequences (Fig. 5B), and transformed into the ign1 plants according to a transformation procedure mediated by Agrobacterium tumefaciens (see "Materials and Methods"). The resultant transgenic plants showed normal growth comparable to wild-type plants under symbiotic conditions and nitrogenase activity of the nodules formed on the transgenic plants was fully recovered to the level of wild-type nodules (Fig. 5C; Table I ). Taking the overall results together, we concluded that the gene encoding an ankyrin-repeat protein is the IGN1 gene.
Genomic Southern hybridization suggested that IGN1 is present as a single copy in the L. japonicus genome (Fig. 6A ). Its transcripts (about 2.3 kb) were detected in flowers, shoots, roots, and nodules at low levels, and the expression levels did not change through nodule development (Fig. 6B).
Structure of the IGN1 Protein
A full-length cDNA for the IGN1 mRNA was amplified by a combination of 5' and 3' RACE procedures from total RNA of nodules (see "Materials and Methods"). Alignment of the cDNA sequence with the genomic sequence defined the structure of the IGN1 gene with three introns (Fig. 7A
). The predicted ORF is 1,788 nucleotides long, which is preceded by a leader sequence of 129 nucleotides and followed by a 3' untranslated region of 320 nucleotides. The conceptual IGN1 protein is 596 amino acids long (MW = 64,510) and contains ankyrin repeats with transmembrane regions (Fig. 7B). The amino terminus is rich in Pro, Glu, Ser, and Thr, as has been described for the PEST sequence that serve as a signal module for conditional or constitutive proteolysis (Rechsteiner and Rogers, 1996
A number of expressed sequence tags from soybean (Glycine max) and M. truncatula were found to share significant similarity with IGN1 at the nucleotide level, suggesting that IGN1 is well conserved in legume plants. A database search using the deduced amino acid sequence of the IGN1 resulted in detection of cDNA clones from Arabidopsis (Arabidopsis thaliana; At3g12360) and Oryza sativa (AK100495), of which the encoded proteins exhibit significant homology with the IGN1 protein. Similarities of their deduced amino acid sequences with the IGN1 protein are 77.2% and 68.3%, respectively. However, physiological functions of these putative IGN1 orthologs are yet to be characterized.
To investigate subcellular localization of IGN1 protein in plant cells we constructed a translational fusion of the IGN1 gene to the green fluorescent protein (GFP) gene. GFP was fused in frame at the N-terminal end of IGN1. The fusion gene was situated downstream of the 35S promoter from Cauliflower mosaic virus, and introduced into tobacco (Nicotiana tabacum) Bright-Yellow 2 (BY2) cells by Agrobacterium-mediated transformation (see "Materials and Methods"). Figure 8
shows GFP fluorescence in the transgenic BY2 cells under a confocal laser-scanning microscope. The fluorescence from the GFP-IGN1 fusion was found only in cell peripherals lining the plasma membrane (Fig. 8, A and D), whereas fluorescence from free GFP was also found in cytoplasm and in the nucleus (Fig. 8G). To verify that the IGN1 fused with GFP resides on the plasma membrane, the transgenic BY2 cells were pulse labeled with FM4-64, which is known as an endocytic tracer translocated from plasma membrane to vacuolar membranes in plant cells (Bolte et al., 2004
To investigate the subcellular localization of IGN1 in the tissues of Lotus plants, we performed immunological detection of IGN1 protein in the membrane fractions prepared from leaves and nodules of L. japonicus (Fig. 9 ). Anti-IGN1 antibody was raised against hexahistidine [(His)6]-tagged polypeptide that corresponds to the ankyrin-repeat moiety of IGN1. The IGN1 protein was clearly detected in the plasma membrane enriched fraction by means of an aqueous two-phase partitioning procedure from leaves and nodules. We further performed another fractionation of nodule cells to obtain PBM enriched fraction by means of disruption of purified symbiosomes (see "Materials and Methods"). An antibody against soybean nodulin 26 as a marker protein for PBM was used to verify the purity of the PBM fraction, indicating that the membrane fraction from purified symbiosomes was highly enriched by PBM. The IGN1 protein was not detectable in the PBM fraction and appeared to be cofractionated with H+-ATPase that serves as a plasma membrane marker. These results indicate that the IGN1 protein in leaves and nodules localizes mainly in the plasma membrane. It should be noted that the fractions from ign1 mutant plants did not show the presence of IGN1, suggesting that the mutants are virtually null for the IGN1 protein.
In this article, we described characterization of a novel Fix– mutant, ign1 of L. japonicus, and molecular identification of the causal gene, IGN1, that encodes an ankyrin-repeat membrane protein. The ign1 mutant is able to form, in early stages of nodule development, apparently normal nodules containing infected cells packed with bacteroids in which nitrogen fixation actually takes place, even though at very low levels. However, the ign1 mutants cannot further develop nitrogen-fixing symbiosis because disintegration of the infected cells occurs just after the onset of nitrogen fixation (Figs. 1 and 3). Thus the ign1 nodules exhibit an obvious early or premature senescence.
A number of Fix– mutants that form ineffective nodules have been isolated from many legume species. Ineffective nodules formed on those Fix– mutants exhibit more or less the phenotype of premature senescence, in both determinate (Imaizumi-Anraku et al., 1997
IGN1 encodes a novel ankyrin-repeat protein with a PEST sequence in the amino terminus and transmembrane domains at the carboxy terminus (Fig. 7). Ankyrin-repeat proteins are widely distributed in prokaryotes, eukaryotes, and some viruses (Sedgwick and Smerdon, 1999
We showed that IGN1 protein is targeted to the plasma membrane when transformed into tobacco BY2 cells as a fusion with GFP (Fig. 8). Furthermore, immunological analysis indicated the localization of IGN1 in plasma membrane fractions isolated from leaves and nodules of L. japonicus (Fig. 9). Localization of IGN1 in PBM that is derived from plasma membrane and serves as the boundary between endosymbionts and the host cell cytoplasm is unlikely because the IGN1 protein could not be detected in the PBM fraction prepared from purified symbiosomes. In contrast, it was clearly detected in the plasma membrane enriched fraction from root nodules. Therefore, we suppose that IGN1 functions in the plasma membrane in nodule cells. Localization in the plasma membrane has been demonstrated for ankyrins in animals, which play a role as an adaptor between cytoskeletal proteins and other proteins such as Na+/K+-ATPase (Denker and Barber, 2002 The ign1 mutant displayed no growth abnormality other than its symbiotic defect. This implies that the IGN1 gene is not crucial for ordinary growth of the plants under nonsymbiotic conditions. Nevertheless, IGN1 is expressed constitutively through all organs of L. japonicus, and is not nodule specific. It is very likely, therefore, that IGN1 makes provision for unfavorable environmental stimuli, such as pathogen attack most probably. In this regard, it will be intriguing to investigate the responses of the ign1 mutant upon infection of pathogenic bacteria and/or fungi. At present, it is too early to assume the exact functions of IGN1 in symbiotic interactions of legume plants with Rhizobium bacteria. However, drastic disorganization of symbiosomes at early stages of nodule formation of the ign1 mutant strongly suggests that IGN1 is required for differentiation and/or persistence of bacteroids and symbiosomes. On the basis of the mutant phenotype and the domain structure of the IGN1 protein, we hypothesize that IGN1 is required for preventing the host plant cells from inappropriately invoking premature senescence, or a kind of defense system, against compatible microsymbionts, thus being essential for functional symbiosis. We will focus our future studies of IGN1 to test this hypothesis.
Plant Materials
A number of symbiotic mutants of Lotus japonicus B-129 Gifu and MG-20 Miyakojima were generated by somatic mutation through intensive culture of calli and/or suspension cells followed by regeneration of the plants (Y. Umehara, unpublished data). The ign1 mutant was isolated by the same method from a progeny of L. japonicus B-129 Gifu transformed with a nodule-specific gene according to a standard hypocotyl transformation procedure (Stiller et al., 1997
Surface-sterilized seeds of L. japonicus B-129 Gifu and of the ign1 mutant were germinated on 0.8% agar plates in an artificially lit growth cabinet on a 16-h-light/8-h-dark cycle at 24°C for 6 d. The seedlings were transplanted onto vermiculite pots supplied with one-half-strength B & D medium with or without nitrogen (Imaizumi-Anraku et al., 1997
An intact plant was placed in a 12 mL glass tube with a rubber cap and incubated in a water bath at 25°C. Acetylene (1 mL) was injected in the tube and the amount of ethylene formed after 15 min was determined by gas chromatography as described (Kouchi et al., 1991
Nodules on root segments were fixed in 0.25% (w/v) glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.2) overnight. Fixed nodules were then sliced at 200 to 250 µm thickness by a razor blade and dehydrated through a graded ethanol series followed by embedding in Teknovit 7100 (Kulzer) according to the manufacturer's instructions. Thin sections (1 µm) were made using an ultramicrotome (Sorvall model MT2-B; DuPont) with a glass knife, attached on a coverslip, and stained with 0.002% (w/v) toluidine blue.
Nodule samples were processed for electron microscopy according to the protocol described by Siddique and Bal (1992)
F2 mapping population was established by crossing the ign1 mutant that was derived from L. japonicus B-129 Gifu with the accession MG-20 Miyakojima (Kawaguchi et al., 2001
Total RNA was prepared by an RNeasy plant mini kit (Qiagen) according to the manufacturer's instructions. Genomic DNA was isolated by the cetyl-trimethyl-ammonium bromide method as previously described (Li et al., 2001 Total RNAs (6 µg) from various organs were subjected to denaturing 1% (w/v) agarose gel electrophoresis. Genomic DNA (3 µg) was digested by restriction enzymes to completion and subjected to 0.7% (w/v) agarose gel electrophoresis. They were transferred onto nylon membranes and hybridized with 32P-labeled IGN1 cDNA probe. A cDNA fragment (416 nucleotides in length) for a portion of IGN1 mRNA was amplified from genomic DNA of wild-type Gifu plants by PCR with a primer set of 5'-GGATCCTCGAGGTGCATACAGAAGCCA-3' (forward) and 5'-ATCGATGGTACCAAGGGATGAACTGAG-3' (reverse) and used to generate the probe with a random prime DNA labeling kit (Takara).
An IGN1 genomic clone was isolated from a genomic library of wild-type Gifu plants prepared in EMBL3 lambda vector (Stratagene) by screening with 32P-labeled IGN1 cDNA as a probe. For the complementation test, an NheI-XbaI fragment (8.6 kb) that contains the entire IGN1 gene was excised, subcloned into a binary vector pCAMBIA1300 (Cambia), and transformed into the ign1 mutant by an Agrobacterium tumefaciens-mediated hypocotyl transformation procedure (Stiller et al., 1997
DNA sequencing was done by the dye-termination method using an automated DNA sequencer (Gene Analyzer 3700, Applied Biosystems). Sequences were analyzed by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and FASTA (http://www.ddbj.nig.ac.jp/search/fasta-j.html). For domain analysis, Pfam (http://www.sanger.ac.uk/Software/Pfam/) was applied. Secondary structures, transmembrane regions, and subcellular targeting peptide were predicted by PredictProtein (http://www.embl-heidelberg.de/predictprotein/predictprotein.html) and TMHMM v2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/), respectively. PEST sequences were defined by PEST-FIND (http://www.at.embnet.org/embnet/tools/bio/PESTfind/).
To generate the GFP-IGN1 fusion construct, a coding region of IGN1 cDNA was amplified with a primer set of 5'-TGTACATGGCTTCCAACTTCAACCAA-3' (forward) and 5'-GCGGCCGCTCAAAGCGCGTAAATTGGAT-3' (reverse) and the amplified fragment was fused to sGFP (S65T) cDNA in frame at the BsrGI site (Niwa et al., 1999
Tobacco (Nicotiana tabacum) BY2 cells were grown in a BY2 medium at 28°C in the dark with continuous shaking (Nagata et al., 1992
Leaves (7 g fresh weight) or nodules (5 g fresh weight) were harvested from L. japonicus and homogenized at 4°C in a mortar with a pestle in 50 mM MOPS-KOH buffer (pH7.5) containing 250 mM Suc, 5 mM EDTA, 5 mM EGTA, 2.5 mM potassium disulfite, 1.5% (w/v) polyvinylpyrrolidone K-30, and 1 mM phenylmethanesulfonyl fluoride. The homogenate was filtered through two layers of Miracloth, centrifuged at 10,000g for 10 min, and the supernatant was further centrifuged at 126,000g for 40 min. The precipitate was washed with 10 mM potassium phosphate and 250 mM Suc (pH 7.8; PS buffer) and resuspended in 1 mL of PS buffer to obtain the microsome fraction, which was further processed by aqueous two-phase partitioning to enrich for plasma membranes (Uemura and Yoshida, 1983
Symbiosomes were prepared from 4-week-old nodules of L. japonicus by the method developed for soybean (Glycine max) nodules (Udvardi et al., 1988
Recombinant IGN1 protein was prepared by a PET system (Novagen). A cDNA fragment corresponding to the N terminus of IGN1 protein including seven ankyrin repeats was PCR amplified from the IGN1 full-length cDNA with a primer pair of 5'-CCATGGCTTCCAACTTCAACCAACC-3' (forward) and 5'-CTCGAGCTCTTTGGAAATATTATGA-3' (reverse). The amplified fragment was digested by NcoI and XhoI, subcloned into expression vector pET21d, and was introduced into Escherichia coli BL21(DE3). An overnight preculture was transferred into Luria-Bertani medium at a 1:10 ratio and incubated at 37°C for 90 min with vigorous shaking. Production of recombinant protein was induced by addition of 1 mM isopropyl D-thiogalactopyranoside and accumulation of expressed protein was achieved by further incubation for 2 h. The cells were harvested by centrifugation, washed with 100 mM Tris-Cl (pH 8.0), and resuspended with the same buffer containing 300 mM NaCl, 1 mM phenylmethanesulfonyl fluoride, and proteinase inhibitor cocktail for His tag (Sigma). The cells were disrupted by sonication and centrifuged at 10,000g for 30 min to remove debris. The supernatant was applied on a nickel-nitrilotriacetic acid agarose (Qiagen) column. The column was washed and developed essentially according to the manufacturer's instructions. The eluate with 100 mM Tris-Cl, 300 mM NaCl, and 400 mM imidazole was diluted in 10 volumes of 100 mM Tris-Cl (pH 8.0) and passed through Q-Sepharose (Amersham) column. The unbound fraction was concentrated by Centricon YM-10 (Millipore) and applied to a Sephacryl S-200 (Amersham) column. The column was developed with 50 mM sodium phosphate and 200 mM NaCl (pH 7.0). The sample after gel filtration was subjected to SDS-PAGE and purified protein with apparent size of 45 kD was detected. This protein was confirmed to be the truncated IGN1 protein by mass spectroscopy. Antiserum against the purified truncated IGN1 protein was raised in rabbit. For affinity purification of the antibody, the antigen was coupled to an NHS-activated Hi-Trap column (Amersham) according to the manufacturer's instructions. The anti-IGN1 antiserum was applied to the antigen column. The column was washed with 50 mM sodium phosphate (pH 7.0) containing 150 mM NaCl and then the bound antibody was eluted with 100 mM Gly-Cl buffer (pH 2.7) followed by immediate neutralization with 1.5 M Tris.
For immunodetection of IGN1 protein, membrane fractions were dissolved in SDS-PAGE sample buffer (62.5 mM Tris-Cl [pH 6.8], 2% [w/v] SDS, 50 mM dithiothreitol, and 8 M urea) and subjected to SDS-PAGE with a 10% (w/v) polyacrylamide gel (Kadenbach et al., 1983
The DNA data bank of Japan accession numbers for IGN1 genomic and cDNA sequences are AB251641 and AB251640, respectively.
We express our sincere thanks to Drs. Masayuki Ishikawa, Yuka Hagiwara, Tetsuo Meshi, and Atsushi Tamai of the National Institute of Agrobiological Sciences for their suggestions and help for expression analysis of GFP-IGN1 fusion protein in BY2 cells. We are also grateful to Dr. Yoshiyuki Tanaka and Ms. Atsuko Nakamura of the National Institute of Agrobiological Sciences for their technical suggestions for aqueous two-phase partitioning. Dr. Hitoshi Sakakibara of Riken Institute and Prof. Dan Roberts of the University of Tennessee are thanked for providing the antibodies against Zea mays H+-ATPase and soybean nodulin 26, respectively. Prof. Robert W. Ridge of International Christian University is thanked for his critical reading of the manuscript. Received December 29, 2006; accepted January 21, 2007; published February 2, 2007.
1 This work was supported by the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, Japan.  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: Hiroshi Kouchi (kouchih{at}nias.affrc.go.jp).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.095356 * Corresponding author; e-mail kouchih{at}nias.affrc.go.jp; fax 81–29–838–8347.
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ABSTRACT
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-Ketoacyl-CoA synthase; II, protein phosphatase 2C; III, IGN1; IV, hypothetical protein; V, pentatricopeptide repeat-containing protein. C, Complementation of the ign1 mutant with an NheI-XbaI fragment containing the complete wild-type IGN1 gene. From left to right: wild type (Gifu), ign1, ign1 transformed with empty vector, ign1 transformed with IGN1 gene. Plants were grown under nitrogen-free conditions and harvested 19 d after inoculation with M. loti. Bar represents 10 mm.
-helices and 
B in animals, are well studied and shown to be involved in transcriptional regulation. I-
