Patent application title: THE AN3 Protein Complex and its Use for Plant Growth Promotion
Geert De Jaeger (Evergem, BE)
Aurine Verkest (Gent, BE)
Dirk Inzé (Moorsel-Aalst, BE)
Dirk Inzé (Moorsel-Aalst, BE)
BASF Plant Science Company GmbH
IPC8 Class: AA01H106FI
Class name: Multicellular living organisms and unmodified parts thereof and related processes method of introducing a polynucleotide molecule into or rearrangement of genetic material within a plant or plant part the polynucleotide alters plant part growth (e.g., stem or tuber length, etc.)
Publication date: 2011-06-30
Patent application number: 20110162110
The present invention relates to an AN3-based protein complex. It relates
further to the use of the complex to promote plant growth, and to a
method for stimulating the complex formation, by overexpressing at least
two member of the complex.
1. An isolated AN3-based protein complex, comprising at least the
proteins AN3p and one or more proteins encoded by AT4G16143, AT1G09270,
AT3G06720, AT5G53480, AT3G60830, AT1G18450, AT2G46020, AT2G28290,
AT1G21700, AT5G14170, AT4G17330, AT4G27550, AT1G65980, AT5G55210,
AT3G15000, AT4G35550, AT1G20670, AT1G08730, AT5G13030, AT2G18876,
AT5G17510, AT1G05370, AT4G21540, AT1G23900 or AT5 G23690.
2. An isolated AN3-based protein complex comprising at least the proteins AN3p and one or more proteins selected from the group consisting of ARP4 (AT1G18450), ARP7 (AT3G60830), SNF2 (AT2G46020), SYD (AT2G28290), SWI3C (AT1G21700) and SWP73B (AT5G14170).
3. The isolated AN3-based protein complex of claim 2, wherein said protein complex comprises at least AN3p, an actin related protein selected from the group consisting of ARP4 and ARP7, an ATPase selected from the group consisting of SNF2 (BRM) and SYD, and a SWIRM domain containing protein.
4. The isolated AN3-based protein complex of claim 3, wherein said SWIRM domain containing protein is SWI3C.
5. A method of promoting plant growth comprising simultaneously overexpressing at least two proteins of the complex of claim 1.
6. A method to promote AN3-based protein complex formation comprising simultaneously ovexpressing at least two proteins of the complex.
 The present invention relates to an AN3-based protein complex. It
relates further to the use of the complex to promote plant growth, and to
a method for stimulating the complex formation, by overexpressing at
least two members of the complex.
 The demand for more plant derived products has spectacularly increased. In the near future the challenge for agriculture will be to fulfill the growing demands for feed and food in a sustainable manner. Moreover plants start to play an important role as energy sources. To cope with these major challenges, a profound increase in plant yield will have to be achieved. Biomass production is a multi-factorial system in which a plethora of processes are fed into the activity of meristems that give rise to new cells, tissues, and organs. Although a considerable amount of research on yield performance is being performed little is known about the molecular networks underpinning yield (Van Camp, 2005). Many genes have been described in Arabidopsis thaliana that, when mutated or ectopically expressed, result in the formation of larger structures, such as leaves or roots. These so-called "intrinsic yield genes" are involved in many different processes whose interrelationship is mostly unknown.
 One of these "intrinsic yield genes", AN3 (also known as GIF1), was identified in search of GRF (growth regulating factor) interactors (Kim and Kende, 2004) and by analysis of narrow-leaf Arabidopsis mutants (Horiguchi et al., 2005). AN3 is a homolog of the human SYT (synovial sarcoma translocation) protein and is encoded by a small gene family in the Arabidopsis genome. SYT is a transcription co-activator whose biological function, despite the implication of its chromosomal translocation in tumorigenesis, is still unclear (Clark et al., 1994; de Bruijn et al., 1996). Using the yeast GAL4 system, AN3 was shown to possess transactivation activity (Kim and Kende, 2004). This together with yeast two-hybrid and in vitro binding assays demonstrating interaction of AN3 with several GRFs (Kim and Kende, 2004; Horiguchi et al., 2005), suggests a role of AN3 as transcription co-activator of GRFs. GRF (growth regulating factor) genes occur in the genomes of all seed plants thus far examined and encode putative transcription factors that play a regulatory role in growth and development of leaves (Kim et al., 2003). In support of a GRF and AN3 transcription activator and co-activator complex, grf and an3 mutants display similar phenotypes, and combinations of grf and an3 mutations showed a cooperative effect (Kim and Kende, 2004). The an3 mutant narrow-leaf phenotype is shown to result of a reduction in cell numbers. Moreover, ectopic expression of AN3 resulted in transgenic plants with larger leaves consisting of more cells, indicating that AN3 controls both cell number and organ size (Horiguchi et al., 2005). Although the function of AN3 in plant growth regulation is not known, these results show that AN3 fulfills the requirements of an "intrinsic yield gene".
 In our ambition to decipher the molecular network underpinning yield enhancement mechanism a genome-wide protein centered approach was undertaken to study AN3 interacting proteins in Arabidopsis thaliana cell suspension cultures. The tandem affinity purification (TAP) technology combined with mass spectrometry (MS) based protein identification resulted in the isolation and identification of 25 AN3 interacting proteins that may function in the regulation of plant growth (Table 2). Surprisingly, we isolated several proteins belonging to multiprotein complexes. Moreover, many interactors are completely uncharacterized. Reports on few of the AN3 interactors show that they are implicated in several developmental processes (Wagner & Meyerowitz, 2002; Meagher et al., 2005; Sarnowski et al., 2005; Hurtado et al., 2006; Kwon et al., 2006) but so far none of the identified genes have been associated with stimulation of plant growth.
 A first aspect of the invention is an isolated AN3-based protein complex, comprising at least the proteins AN3p and one or more of the proteins selected from the group encoded by AT4G16143, AT1G09270, AT3G06720, AT5G53480, AT3G60830, AT1G18450, AT2G46020, AT2G28290, AT1G21700, AT5G14170, AT4G17330, AT4G27550, AT1G65980, AT5G55210, AT3G15000, AT4G35550, AT1G20670, AT1G08730, AT5G13030, AT2G 18876, AT5G17510, AT1G05370, AT4G21540, AT1G23900 and AT5G23690 (genes listed in Table II). Preferably, said AN3-based protein complex comprises at least the proteins AN3p and one or more proteins selected from the group consisting of ARP4 (AT1G18450), ARP7 (AT3G60830), SNF2 (AT2G46020), SYD (AT2G28290), SWI3C (AT1G21700) and SWP73B (AT5G14170). Even more preferably, said AN3-based protein complex comprises at least AN3p, an actin related protein selected from the group consisting of ARP4 and ARP7, an ATPase selected from the group consisting of SNF2 (BRM) and SYD and a SWIRM domain containing protein. Preferably, said SWIRM domain containing protein is SWI3C. An AN3-based protein complex as used here means that AN3p is interacting, directly or indirectly, with the other proteins of the complex. A direct interaction is an interaction where at least one domain of AN3p interacts with one or more domains or the interaction partner. An indirect interaction is an interaction where AN3p itself is not interacting with the interacting protein by one of its domains, but where said interacting protein is interacting with a protein that is directly or indirectly interacting with AN3p.
 A further aspect of the invention is the use of a protein complex according to the invention to promote plant growth. Preferably, said use is an overexpression of the protein complex, by overexpressing at least two members of the protein complex. Promotion of plant growth, as used here, is an increase in plant biomass in plants where the protein complex is used, compared with the same plant where the complex is not used, grown under the same conditions, except for the conditions needed for the use of the complex, if any. Such conditions may be, as a non limited example, the addition of one or more compounds to induce one or more promoters of one or more genes encoding a protein of the complex. Alternatively, the same plant is an untransformed parental plant, grown under the same conditions as the transformed plant, wherein the complex is used. Preferably, promotion of plant growth results in an increased yield. This yield can be a total increase in plant biomass, or a partial increase of yield, such as, but not limited to seed yield, leave yield or root yield.
 Still another aspect of the invention is a method to promote AN3-based protein complex formation, by simultaneous overexpression of at least two proteins of the complex. Proteins of the complex, beside AN3p itself, are listed in table II. Preferably, said overexpression is an overexpression of AN3p and one or more proteins selected from the group consisting of ARP4 (AT1G18450), ARP7 (AT3G60830), SNF2 (AT2G46020), SYD (AT2G28290), SWI3C (AT1G21700) and SWP73B (AT5G14170). Even more preferably, said overexpression is an overexpression of at least AN3p, an actin related protein selected from the group consisting of ARP4 and ARP7, an ATPase selected from the group consisting of SNF2 (BRM) and SYD and a SWIRM domain containing protein. Preferably, said SWIRM domain containing protein is SWI3C.
 Methods for obtaining overexpression are known to the person skilled in the art, and comprise, but are not limited to placing the gene encoding the protein to be overexpressed after a strong promoter such as the Cauliflower Mosaic Virus 35S promoter. Simultaneous overexpression as used here means that there is an overlap in timeframe for all the proteins to be overexpressed, whereby the level of said proteins is increased when compared to a non-overexpressed control. It does not necessarily mean that all genes should be induced at the same moment. Depending upon the turnover of the messenger RNA and/or the protein, one gene may be induced before or after another, as long as there is an overlap in time where both proteins are present in a concentration that is higher than the normal (non-overexpressed) concentration.
BRIEF DESCRIPTION OF THE FIGURES
 FIG. 1. Expression analysis of GS-tagged GFP and AN3 in transgenic cell suspension cultures.
 The total protein extract of 2-day-old wild-type and N- and C-terminal GS-tagged GFP and AN3 overexpressing cultures (60 μg) was separated by 12% SDS-PAGE and immunoblotted. For detection of GS-tagged proteins, blots were incubated with human blood plasma followed by incubation with anti-human IgG coupled to horseradish peroxidase. Protein gel blots were developed by Chemiluminiscent detection. The expected recombinant molecular masses for GS-tagged GFP and AN3 are 52.8 kDa and 43.5 kDa, respectively (indicated with a black dot).
 FIG. 2. Analysis of the TAP protein eluates.
 GS-tagged protein complexes were purified from transgenic plant cell suspension cultures, precipitated with TCA (25%, v/v), separated on 4-12% NuPAGE gels, and visualized with colloidal Coomassie G-250 staining. Bait proteins are indicated with a dot.
Materials and Methods to the Examples
 Construction of N- and C-terminal GS-tagged GFP and AN3 under the control of the 35S (CaMV) promoter was obtained by Multisite Gateway LR reactions. The coding regions, without (-) and with (+) stopcodon, were amplified by polymerase chain reaction (PCR) and cloned into the Gateway pDONR221 vector (Invitrogen) resulting in pEntryL1L2-GFP(-) pEntryL1L2-GFP(+), pEntryL1L2-AN3(-) and pEntryL1L2-AN3(+). The Pro35S:GFP-GS- and Pro35S:AN3-GS-containing plant transformation vectors were obtained by Multisite Gateway LR reaction between pEntryL4R1-Pro35S, pEntryL1L2-GFP(-) or pEntryL1L2-AN3(-), and pEntryR2L3-GS and the destination vector pKCTAP, respectively (Van Leene et al., 2007). To obtain the Pro35S:GS-GFP and Pro35S:GS-AN3 vectors Multisite LR recombination between pEntryL4L3-Pro35S and pEntryL1L2-GFP(+) or pEntryL1L2-AN3(+) with pKNGSTAP occurred.
 All entry and destination vectors were checked by sequence analysis. Expression vectors were transformed to Agrobacterium tumefaciens strain C58C1 RifR (pMP90) by electroporation. Transformed bacteria were selected on yeast extract broth plates containing 100 μg/mL rifampicin, 40 μg/mL gentamicin, and 100 μg/mL spectinomycin.
Cell Suspension Cultivation
 Wild-type and transgenic Arabidopsis thaliana cell suspension PSB-D cultures were maintained in 50 mL MSMO medium (4.43 g/L MSMO, Sigma-Aldrich), 30 g/L sucrose, 0.5 mg/L NAA, 0.05 mg/L kinetin, pH 5.7 adjusted with 1M KOH) at 25° C. in the dark, by gentle agitation (130 rpm). Every 7 days the cells were subcultured in fresh medium at a 1/10 dilution.
Cell Culture Transformation
 The Arabidopsis culture was transformed by Agrobacterium co-cultivation as described previously (Van Leene et al., 2007). The Agrobacterium culture exponentially growing in YEB (OD600 between 1.0 and 1.5) was washed three times by centrifugation (10 min at 5000 rpm) with an equal volume MSMO medium and resuspended in cell suspension growing medium until an OD600 of 1.0. Two days after subcultivation, 3 mL suspension culture was incubated with 200 μL washed Agrobacteria and 200 μM acetoseringone, for 48 h in the dark at 25° C. with gentle agitation (130 rpm). Two days after co-cultivation, 7 mL MSMO containing a mix of three antibiotics (25 μg/mL kanamycin, 500 μg/mL carbenicellin, and 500 μg/mL vancomycin) was added to the cell cultures and grown further in suspension under standard conditions (25° C., 130 rpm and continuous darkness). The stable transgenic cultures were selected by sequentional dilution in a 1:5 and 1:10 ratio in 50 mL fresh MSMO medium containing the antibiotics mix, respectively at 11, and 18 days post co-cultivation. After counter selecting the bacteria, the transgenic plant cells were further subcultured weekly in a 1:5 ratio in 50 mL MSMO medium containing 25 μg/mL kanamycin for two more weeks. Thereafter the cells were weekly subcultured in fresh medium at a 1/10 dilution.
Expression Analysis of Cell Suspension Cultures
 Transgene expression was analyzed in a total protein extract derived from exponentially growing cells, harvested two days after subculturing. Equal amounts of total protein were separated on 12% SDS-PAGE gels and blotted onto Immobilon-P membranes (Millipore, Bedford, Mass.). Protein gel blots were blocked in 3% skim milk in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Triton X-100. For detection of GS-tagged proteins, blots were incubated with human blood plasma followed by incubation with anti-human IgG coupled to horseradish peroxidase (HRP; GE-Healthcare). Protein gel blots were developed by Chemiluminiscent detection (Perkin Elmer, Norwalk, Conn.).
Protein Extract Preparation
 Cell material (15 g) was grinded to homogeneity in liquid nitrogen. Crude protein extract were prepared in an equal volume (w/v) of extraction buffer (25 mM Tris-HCl, pH 7.6, 15 mM MgCl2, 5 mM EGTA, 150 mM NaCl, 15 mM p-nitrophenylphosphate, 60 mM β-glycerophosphate, 0.1% (v/v) Nonidet P-40 (NP-40), 0.1 mM sodium vanadate, 1 mM NaF, 1 mM DTT, 1 mM PMSF, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 5 μg/mL antipain, 5 μg/mL chymostatin, 5 μg/mL pepstatin, 10 μg/mL soybean trypsin inhibitor, 0.1 mM benzamidine, 1 μM trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), 5% (v/v) ethylene glycol) using an Ultra-Turrax T25 mixer (IKA Works, Wilmington, N.C.) at 4° C. The soluble protein fraction was obtained by a two-step centrifugation at 36900 g for 20 min and at 178000 g for 45 min, at 4° C. The extract was passed through a 0.45 μm filter (Alltech, Deerfield, Ill.) and the protein content was determined with the Protein Assay kit (Bio-Rad, Hercules, Calif.).
Tandem Affinity Purification
 Purifications were performed as described by Burckstummer et al. (2006), with some modifications. Briefly, 200 mg total protein extract was incubated for 1 h at 4° C. under gentle rotation with 100 μL IgG Sepharose 6 Fast Flow Flow beads (GE-Healthcare, Little Chalfont, UK), pre-equilibrated with 3 mL extraction buffer. The IgG Sepharose beads were transferred to a 1 mL Mobicol column (MoBiTec, Goettingen, Germany) and washed with 10 mL IgG wash buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% NP-40, 5% ethylene glycol) and 5 mL Tobacco (Nicotiana tabacum L.) Etch Virus (TEV) buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% (v/v) NP-40, 0.5 mM EDTA, 1 mM PMSF, 1 μM E64, 5% (v/v) ethylene glycol). Bound complexes were eluted via AcTEV digest (2×100U, Invitrogen) for 1 h at 16° C. The IgG eluted fraction was incubated for 1 h at 4° C. under gentle rotation with 100 μL Streptavidin resin (Stratagene, La Jolla, Calif.), pre-equilibrated with 3 mL TEV buffer. The Streptavidin beads were packed in a Mobicol column, and washed with 10 mL TEV buffer. Bound complexes were eluted with 1 mL streptavidin elution buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% (v/v) NP-40, 0.5 mM EDTA, 1 mM PMSF, 1 μM E64, 5% (v/v) ethylene glycol, 20 mM Desthiobiotin), and precipitated using TCA (25% v/v). The protein pellet was washed twice with ice-cold aceton containing 50 mM HCl, redissolved in sample buffer and separated on 4-12% gradient NuPAGE gels (Invitrogen). Proteins were visualized with colloidal Coomassie brilliant blue staining.
Proteolysis and Peptide Isolation
 After destaining, gel slabs were washed for 1 hour in H2O, polypeptide disulfide bridges were reduced for 40 min in 25 mL of 6.66 mM DTT in 50 mM NH4HCO3 and sequentially the thiol groups were alkylated for 30 min in 25 mL 55 mM IAM in 50 mM NH4HCO3. After washing the gel slabs 3 times with water, complete lanes from the protein gels were cut into slices, collected in microtiter plates and treated essentially as described before with minor modifications (Van Leene et al., 2007). Per microtiterplate well, dehydrated gel particles were rehydrated in 20 μL digest buffer containing 250 ng trypsin (MS Gold; Promega, Madison, Wis.), 50 mM NH4HCO3 and 10% CH3CN (v/v) for 30 min at 4° C. After adding 10 μL of a buffer containing 50 mM NH4HCO3 and 10% CH3CN (v/v), proteins were digested at 37° C. for 3 hours. The resulting peptides were concentrated and desalted with microcolumn solid phase tips (PerfectPure® C18 tip, 200 nL bed volume; Eppendorf, Hamburg, Germany) and eluted directly onto a MALDI target plate (Opti-TOF® 384 Well Insert; Applied Biosystems, Foster City, Calif.) using 1.2 μL of 50% CH3CN: 0.1% CF3COOH solution saturated with α-cyano-4-hydroxycinnamic acid and spiked with 20 fmole/μL Glu1-Fibrinopeptide B (Sigma-Aldrich), 20 fmole/μL des-Pro2-Bradykinin (Sigma-Aldrich), and 20 fmole/μL Adrenocorticotropic Hormone Fragment 18-39 human (Sigma-Aldrich).
Acquisition of Mass Spectra
 A MALDI-tandem MS instrument (4800 Proteomics Analyzer; Applied Biosystems) was used to acquire peptide mass fingerprints and subsequent 1 kV CID fragmentation spectra of selected peptides. Peptide mass spectra and peptide sequence spectra were obtained using the settings essentially as presented in Van Leene et al. (2007). Each MALDI plate was calibrated according to the manufacturers' specifications. All peptide mass fingerprinting (PMF) spectra were internally calibrated with three internal standards at m/z 963.516 (des-Pro2-Bradykinin), m/z 1570.677 (Glu1-Fibrinopeptide B), and m/z 2465,198 (Adrenocorticotropic Hormone Fragment 18-39) resulting in an average mass accuracy of 5 ppm±10 ppm for each analyzed peptide spot on the analyzed MALDI targets. Using the individual PMF spectra, up to sixteen peptides, exceeding a signal-to-noise ratio of 20 that passed through a mass exclusion filter were submitted to fragmentation analysis.
MS-Based Protein Homology Identification
 PMF spectra and the peptide sequence spectra of each sample were processed using the accompanied software suite (GPS Explorer 3.6, Applied Biosystems) with parameter settings essentially as described in Van Leene et al. (2007). Data search files were generated and submitted for protein homology identification by using a local database search engine (Mascot 2.1, Matrix Science). An in-house nonredundant Arabidopsis protein database called SNAPS Arabidopsis thaliana version 0.4 (SNAPS=Simple Nonredundant Assembly of Protein Sequences, 77488 sequence entries, 30468560 residues; available at http://www.ptools.ua.ac.be/snaps) was compiled from nine public databases. Protein homology identifications of the top hit (first rank) with a relative score exceeding 95% probability were retained. Additional positive identifications (second rank and more) were retained when the score exceeded the 98% probability threshold.
Expression Analysis of GS-Tagged GFP and AN3 Overexpressing Cell Lines
 Before performing TAP purifications stably transformed cell suspension cultures were screened on the protein expression level of the transgenes. Protein gel blotting of equal amounts of total protein extract derived from wild-type (PSB-D) cultures and GS-GFP, GFP-GS, GS-AN3, and AN3-GS overexpressing cell lines showed clear expression of the GS-tagged proteins (FIG. 1).
TAP Purification of Wild-Type and GS-Tagged GFP Overexpressing Cultures
 Despite the two successive purification steps performed within TAP purifications, background proteins co-purified by non-specific binding are an issue. Contaminating proteins due to experimental background were determined by purifications on wild-type and transgenic cultures overexpressing N- and C-terminal GS-tagged nuclear localized green fluorescent protein (GFP). Non-specific co-purified proteins were precipitated, separated on gel, stained (FIG. 2), trypsin digested and identified unambiguously by MALDI-TOF/TOF. Most contaminants are high abundant proteins, such as chaperones, cytoskeleton proteins, ribosomal proteins, metabolic enzymes, or protein translation factors (Table 1). Identical or similar proteins were found as common contaminants in other plant protein-protein interaction studies (Rohila et al., 2006; Van Leene et al., 2007).
TAP Isolation and MS Identification of AN3 Interacting Proteins
 In order to identify the interaction partners of AN3 in vivo, we performed tandem affinity (TAP) purifications on N- and C-terminal GS-fusions of AN3 ectopically expressed under control of the constitutive 35SCaMV promoter in transgenic Arabidopsis suspension cultures. Two independent TAP purifications were performed on extracts from AN3-GS and GS-AN3 lines, harvested two days after sub-culturing into fresh medium. The affinity purified proteins were separated on a 4-12% NuPAGE gel and stained with Coomassie Brilliant Blue. The purification profiles from transgenic cultures overexpressing AN3 is shown in FIG. 2. Protein bands were cut, in-gel digested with trypsin and subjected to MALDI-TOF/TOF mass spectrometry for protein identification. After substracting background proteins, identified by the control purifications described in example 2 and in other analyses (GUS and cytosolic GFP, Van Leene et al., 2007), from the obtained hit list we identified 25 AN3 interacting proteins (Table 2). These can be divided into two groups: 14 proteins were confirmed experimentally and 11 proteins were identified only in one out of four TAP experiments.
Isolation and Subunit Identification of AN3 Interacting SWI/SNF Chromatin Remodeling Complexes in Plants
 Among the experimentally confirmed AN3 interactors six proteins act as subunits of macromolecular machines that remodel chromatin structure. A database survey (ChromDB, Gendler et al., 2008) illustrates that all of them belong to the SWI/SNF ATPase family. SWI/SNF chromatin remodeling ATPases are conserved in the animal and the plant kingdom and regulate transcriptional programs in response to endogenous and exogenous cues. This suggests that the transcriptional activity of AN3 is regulated through chromatin remodeling. In agreement, the human AN3 homolog SYT was also shown to interact with the SWI/SNF complex components BRM and Brg1 (Thaete et al., 1999; Perani et al., 2003; Ishida et al., 2004).
 Although the functional role of several putative SWI/SNF complex components has been studied in Arabidopsis, so far no complete plant chromatin remodeling complex has been isolated and characterized. The co-purification with AN3 gives for the first time prove of the in vivo physical composition of plant SWI/SNF complexes which before was based solely on homology analyses and the interpretation of genetic and in vitro interactions. A literature survey illustrates that SWI/SNF ATPase subunits control multiple developmental pathways in Arabidopsis. Null mutants of the two isolated ATPases SYD (At2g28290) and BRM (SNF2) (At2g46020) display pleiotropic developmental defects. Both mutants are slow growing and dwarfed, have defects in cotyledon separation, and exhibit reduced apical dominance (Wagner & Meyerowitz, 2002; Farrona et al., 2004; Hurtado et al., 2006; Kwon et al., 2006; Su et al., 2006). Null mutants in BRM (SNF2) also have unique root growth defects and are male sterile (Wagner & Meyerowitz, 2002; Hurtado et al., 2006; Kwon et al., 2006). Core complex Swi3c (At1g21700) mutants closely resemble brm mutants (Sarnowski et al., 2005). Mutants of the accessory components ARP4 and ARP7 display pleiotropic defects with less resemblance to the syd, brm and swi3c phenotypes (Meagher et al., 2005). Down-regulation of ARP4 resulted in phenotypes including altered organization of plant organs, early flowering, delayed flower senescence and partial sterility (Kandasamy et al., 2005a). ARP7 knockdown results in dwarfed plants with small rosette leaves, highly retarded root growth, altered flower development and reduced fertility (Kandasamy et al., 2005b). Finally, RNAi-mediated silencing of the accessory SWI/SNF complex component SWP73B (At5g14170) resulted in dwarfed plants with shorter roots (Crane & Gelvin, 2007).
Isolation and Identification of AN3 Interactors
 With the exception of the SWI/SNF chromatin remodeling complex subunits all other 19 identified AN3 interactors are not or poorly characterized. Table 3 gives an overview of there GO biological process and molecular function.
 Among them four interactors (At4g16143, At1g09270, At3g06720 and At5g53480) are involved in nucleocytoplasmic trafficking which identifies AN3 as one of the targets of plant nuclear transporters. Indeed a precise cellular localization is essential for protein function and nuclear localization is a key to the function of transcription factors. In plants, nucleocytoplasmic trafficking plays a critical role in various biological processes (Meier, 2007; Xu & Meier, 2008) and nuclear transporters have been shown to be involved in regulating different signal transduction pathways during plant development (Bollman et al., 2003) and in plant responses to biotic (Palma et al., 2005) and abiotic stresses (Verslues et al., 2006).
 Another AN3 interactor, that is yet not characterized, is the trehalose phosphatase/synthase 4 (TPS4). Several studies in plants imply an important role of trehalose biosynthesis for plant growth, development and stress tolerance (Grennan, 2007). In the case of Arabidopsis TPS1, knockout mutants display an embryo lethal phenotype, suggesting a role of this gene in plant development (Eastmond et al., 2002). In addition, overexpression of TPS1 shed light on its role as a regulator of glucose, abscisic acid, and stress signalling (Avonce et al., 2004). The latter study, together with a recent analysis of a rice TPS triggering abiotic stress response gene induction when overexpressed (Ge et al., 2008), suggests a possible role for TPS genes in regulating transcriptional signaling pathways.
 The other identified interactors indicate links of AN3 function in multiple processes. Several studies demonstrate the involvement of sphingosine kinases in plant cell signaling (Coursol et al., 2003; Coursol et al., 2005; Worral et al., 2008), whereas reports on myosin homologues (Peremyslov et al., 2008; Jiang et al., 2007) implicate roles of protein and organelle trafficking in plant development. The connections between these genes, the other identified interactors and AN3 will be interesting to study in the future.
TABLE-US-00001 TABLE 1 List of co-purifying proteins during TAP experiments of untransformed cell cultures, and of cultures ectopically expressing nuclear localized GFP Accession number Protein name Mock GFP At1g06780 glycosyl transferase family 8 protein + At1g07930 elongation factor 1-alpha + At1g09080 luminal binding protein 3 (BiP-3) (BP3) + At1g13440 glyceraldehyde 3-phosphate dehydrogenase, cytosolic, + At1g31230 bifunctional aspartate kinase/homoserine dehydrogenase + At1g34610 Ulp1 protease family protein + At1g50010 tubulin alpha chain + At1g61210 WD-40 repeat family protein/katanin p80 subunit, putative + At1g75010 MORN repeat-containing protein + At1g79920 heat shock protein 70, putative + At1g79930 heat shock protein, putative + At2g07620 putative helicase + At2g21410 vacuolar proton ATPase, putative + At2g26570 expressed protein + At3g07160 glycosyl transferase family 48 protein + At3g09170 Ulp1 protease family protein + At3g09440 heat shock cognate 70 kDa protein 3 + At3g11950 ATHST; prenyltransferase + At3g12580 heat shock protein 70, putative + At3g17390 S-adenosylmethionine synthetase, putative + At3g18530 expressed protein + At3g26020 serine/threonine protein phosphatase 2A regulatory subunit B' + At3g42100 AT hook motif-containing protein-related + At3g48870 ATP-dependent Clp protease ATP-binding subunit (ClpC) + At3g49640 nitrogen regulation family protein + At3g54940 cysteine proteinase, putative + At4g00020 BRCA2A (breast cancer 2 like 2A) + At4g09800 40S ribosomal protein S18 + At4g14960 tubulin alpha chain + At4g18080 hypothetical protein + At4g20160 expressed protein + At4g20890 tubulin beta chain + At4g31820 phototropic-responsive NPH3 family protein + At4g33200 myosin, putative + At5g02490 heat shock cognate 70 kDa protein 2 + At5g02500 heat shock cognate 70 kDa protein 1 + At5g08670 ATP synthase beta chain, mitochondrial + At5g08680 ATP synthase beta chain, mitochondrial + At5g08690 ATP synthase beta chain, mitochondrial + At5g09810 actin 7 (ACT7)/actin 2 + + At5g18110 Novel cap-binding protein (nCBP) + At5g28540 luminal binding protein 1 (BiP-1) (BP1) + + At5g35360 acetyl-CoA carboxylase, biotin carboxylase subunit (CAC2) + At5g40060 disease resistance protein (TIR-NBS-LRR class), putative + At5g42020 luminal binding protein 2 (BiP-2) (BP2) + At5g44340 tubulin beta chain + At5g60390 elongation factor 1-alpha + At5g62700 tubulin beta chain +
TABLE-US-00002 TABLE 2 List of AN3-copurified proteins identified by MS. The last column tells in how many of the four independent experiments an interactor was identified. Protein Best ion MW Peptide score/ score/ AGI code Description (kDa) count threshold threshold AT4G16143 importin alpha-2, putative (IMPA2) 49.5 13 388/61 84/28 2 AT1G09270 importin alpha-1 subunit, putative (IMPA4) 59.4 6 74/61 37/31 1 AT3G06720 importin alpha-1 subunit, putative (IMPA1) 58.6 8 160/61 62/28 2 AT5G53480 importin beta-2, putative 96.2 16 295/61 50/32 2 AT3G60830 actin-related protein 7 (ARP7) 39.9 12 285/61 53/28 3 AT1G18450 actin-related protein 4 (ARP4) 48.9 12 230/61 44/28 2 AT2G46020 transcription regulatory protein SNF2 (ATPase) 245.4 31 351/61 57/31 2 AT2G28290 chromatin remodeling protein, SYD ATPase 389.8 22 118/61 53/31 4 AT1G21700 SWIRM domain-containing protein/DNA-binding family protein 88.2 5 32/32 2 AT5G14170 SWIB complex BAF60b domain-containing protein 59.2 18 302/61 43/31 2 AT4G17330 G2484-1, agenet (tudor-like) domain-containing protein 113.3 25 317/61 61/32 3 AT4G27550 trehalose phosphatase/synthase 4 89.4 15 68/61 2 AT1G65980 thioredoxin-dependent peroxidase 17.4 8 80/61 2 AT5G55210 expressed protein 18.5 4 105/61 49/31 2 AT3G15000 expressed protein similar to DAG protein 42.8 3 38/30 2 AT4G35550 homeobox-leucine zipper protein (HB-2)/HD-ZIP protein 29.6 3 33/28 1 AT1G20670 DNA-binding bromodomain-containing protein 72.9 16 75/61 1 AT1G08730 myosin heavy chain (PCR43) (Fragment) 174.6 18 70/61 1 AT5G13030 expressed protein 71.1 3 31/29 1 AT2G18876 expressed protein 43.5 11 67/61 1 AT5G17510 expressed protein 42.5 3 37/28 1 AT1G05370 expressed protein 49.9 12 66/61 1 AT4G21540 putative sphingosine kinase (SphK) 141.7 9 69/61 1 AT1G23900 gamma-adaptin 96.4 19 78/61 1 AT5G23690 polynucleotide adenylyltransferase family protein 59.6 11 66/61 1
TABLE-US-00003 TABLE 3 AGI Code Name/Description GO Biological Process GO Molecular Function At4g16143 Importin alpha-2 (IMP2) Protein import into nucleus Protein transporter activity At1g09270 Importin alpha-1 (IMPA4) Intracellular protein transport Protein transporter activity At3g06720 Importin alpha-1 (IMPA1) Intracellular protein transport Protein transporter activity At5g53480 Importin beta-2 Protein import into nucleus Protein transporter activity At4g17330 G2484-1 protein unknown RNA binding At4g27550 Trehalose phosphatase/synthase 4 Trehalose biosynthesis Trehalose phosphate (TPS4) synthase activity At1g65980 Thioredoxin-dependent peroxidase 1 unknown Antioxidant activity (TPX1) At5g55210 Expressed protein unknown unknown At3g15000 Expressed protein similar to DAG unknown unknown protein At4g35550 Wuschel-related homeobox 13 Regulation of transcription DNA binding (WOX13) At1g20670 Bromodomain-containing protein unknown DNA binding At1g08730 Myosin-like protein XIC Actin filament-based movement Protein binding At5g13030 Expressed protein unknown unknown At2g18876 Expressed protein unknown unknown At5g17510 Expressed protein unknown unknown At1g05370 Expressed protein unknown unknown At4g21540 Putative sphingosine kinase Activation of protein kinase C Kinase activity activity At1g23900 Gamma-adaptin Vesicle-mediated transport Clathrin binding At5g23690 Polynucleotide adenylyltransferase RNA processing RNA binding protein
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